Team:OU-Norman OK/Project/Notebook

From 2013.igem.org

(Difference between revisions)

Latest revision as of 04:17, 28 September 2013

09/13/13

Gel of Pptb-

Row 1
Lane Number	Sample
1	Ladder (1kb plus)
2	Pptb- #9A
3	Pptb- #9 B
4	Empty
5	Empty
6	Empty
7	Empty
8	Empty

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09/13/13

Digest of Pptb- Sample

500ng X (μL/178.1ng) = 2.81μL

500ng X (μL/110.1ng) = 4.54μL

Sample	EcoR1	Pst1	10x Buffer	DNA	PCR dH₂O	Total
Pptb- #9A	2μL	2μL	2μL	2.81μL	11.19μL	20μL
Pptb- #9B	2μL	2μL	2μL	4.54μL	9.46μL	20μL

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09/11/13

Quantification and DNA Extraction of 3A

QIAprep Spin Miniprep Kit (250)

HTML tutorial

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09/10/13

Gel of Pptb- + Clos Ori

Row #1
Lane Number	Sample
1	Ladder (1kb plus)
2	Pptb- #1
3	pptb- #2
4	Pptb- #3
5	Pptb- #4
6	Pptb- #6
7	Empty
8	Empty

HTML tutorial

Expected insert size of 125bp

No valid inserts found

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09/11/13

Digest of Pptb-

Pptb- #1

500ng X (μL/81.1ng) = 6.17μL

Pptb- #2

500ng X (μL/86.4ng) = 5.79μL

Pptb- #3

500ng X (μL/70.9ng) = 7.05μL

Pptb- #4

500ng X (μL/147.7ng) = 3.39μL

Pptb- #6

500ng X (μL/169.3ng) = 2.95μL

Sample	EcoR1	Pst1	10x Buffer	DNA	PCR dH₂O	Total
Pptb- #1	2μL	2μL	2μL	6.17μL	9.83μL	20μL
Pptb- #2	2μL	2μL	2μL	5.79μL	8.21μL	20μL
Pptb- #3	2μL	2μL	2μL	7.05μL	6.95μL	20μL
Pptb- #4	2μL	2μL	2μL	3.39μL	10.61μL	20μL
Pptb- #5	2μL	2μL	2μL	2.95μL	11.05μL	20μL

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09/11/13

Quantification and DNA Extraction of 3A

QIAprep Spin Miniprep Kit (250)
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09/10/13

Gel of Pptb- + Clos Ori

Row #1
Lane Number	Sample
1	Ladder (1kb plus)
2	Pptb- #1
3	pptb- #2
4	Pptb- #3
5	Pptb- #4
6	Pptb- #5
7	Pptb- #7
8	Pptb- #14
9	Pptb- #15
10	Empty
11	Empty
12	Empty
13	Empty
14	Empty
15	EMpty
16	Empty
Row #2
Lane Number	Sample
1	Ladder (1kb plus)
2	Pptb- #16
3	Pptb- #17
4	Pptb- #18
5	Pptb- #21
6	Pptb- #22
7	Pptb- #23
8	Pptb- #24
9	Pptb- #25
10	Empty
11	Empty
12	Empty
13	Empty
14	Empty
15	EMpty
16	Empty

HTML tutorial

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09/6/13

Digest of Pptb- and Clos Ori

3 #17

500ng X (μL/123.2ng) = 4.06μL

Pptb- #1

500ng X (μL/104.6ng) = 4.78μL

Pptb- #2

500ng X (μL/123.1ng) = 4.06μL

Pptb- #3

500ng X (μL/92.7ng) = 5,39μL

Pptb- #4

500ng X (μL/74.5ng) = 6.71μL

Pptb- #5

500ng X (μL/114.6ng) = 4.36μL

Pptb- #7

500ng X (μL/154.8ng) = 3.23μL

Pptb- #14

500ng X (μL/127.9ng) = 3.91μL

Pptb- #15

500ng X (μL/110.4ng) = 4.53μL

Pptb- #16

500ng X (μL/112.4ng) = 4.45μL

Pptb- #17

500ng X (μL/124.5ng) = 4.01μL

Pptb- #18

500ng X (μL/219.8ng) = 2.27μL

Pptb- #21

500ng X (μL/157.6ng) = 3.17μL

Pptb- #22

500ng X (μL/145.6ng) = 3.43μL

Pptb- #23

500ng X (μL/134.2ng) = 3.73μL

Pptb- #24

500ng X (μL/114.6ng) = 4.36μL

Pptb- #25

500ng X (μL/189.8ng) = 2.63μL

Sample	EcoR1	Pst1	10x Buffer	DNA	PCR dH₂O	Total
Pptb- #1	2μL	2μL	2μL	4.78μL	9.22μL	20μL
Pptb- #2	2μL	2μL	2μL	4.06μL	9.94μL	20μL
Pptb- #3	2μL	2μL	2μL	5.39μL	8.61μL	20μL
Pptb- #4	2μL	2μL	2μL	6.71μL	7.29μL	20μL
Pptb- #5	2μL	2μL	2μL	4.36μL	9.64μL	20μL
Pptb- #7	2μL	2μL	2μL	3.23μL	10.77μL	20μL
Pptb- #14	2μL	2μL	2μL	3.91μL	10.09μL	20μL
Pptb- #815	2μL	2μL	2μL	4.53μL	9.47μL	20μL
Pptb- #16	2μL	2μL	2μL	4.45μL	9.55μL	20μL
Pptb- #17	2μL	2μL	2μL	4.02μL	9.98μL	20μL
Pptb- #18	2μL	2μL	2μL	2.27μL	11.73μL	20μL
Pptb- #21	2μL	2μL	2μL	3.17μL	10.83μL	20μL
Pptb- #22	2μL	2μL	2μL	3.43μL	10.57μL	20μL
Pptb- #23	2μL	2μL	2μL	3.73μL	10.27μL	20μL
Pptb- #24	2μL	2μL	2μL	4.36μL	9.64μL	20μL
Pptb- #25	2μL	2μL	2μL	2.63μL	11.37μL	20μL

Heat shock for 45 minutes in water bath and 15 minutes at 80°C on heat block to inactivate EcoR1

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09/6/13

Quantification and DNA Extraction of 3A

QIAprep Spin Miniprep Kit (250)

HTML tutorial

" alt="HTML tutorial" width="300" height="300" >

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09/5/13

Gel of Plep #1

Row 1
Lane Number	Sample
1	Ladder (1kb plus)
2	Plep #1
3	Empty
4	Empty
5	Empty
6	Empty
7	Empty
8	Empty

HTML tutorial

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09/5/13

Digest of Plep #1

Amount	Component
6.67μL	Plep
2μL	10x Buffer
2μL	EcoR1
2μL	Pst1
7.33μL	PCR Water

500ng X (μL/75ng) = 6.67μL

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09/5/13

Quantification and DNA Extraction of 3A

QIAprep Spin Miniprep Kit (250)

HTML tutorial

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09/4/13

Gel of Colony PCR

Row #1
Lane Number	Sample
1	Ladder (1kb plus)
2	T2 #1
3	T2 #2
4	T2 #3
5	T2 #4
6	Ter #5
7	T2 #16
8	T2 #17
9	T2 #18
10	T2 #19
11	T2 #20
12	Empty
13	Empty
14	Empty
15	EMpty
16	Empty
Row #2
Lane Number	Sample
1	Ladder (1kb plus)
2	Pveg2 #1
3	Pveg2 #2
4	Pveg2 #3
5	Pveg2 #4
6	Pveg2 #5
7	Pveg2 #16
8	Pveg2 #17
9	Pveg2 #18
10	Pveg2 #19
11	Pveg2 #20
12	Empty
13	Empty
14	Empty
15	EMpty
16	Empty

HTML tutorial

Row #1
Lane Number	Sample
1	Ladder (1kb plus)
2	Pptb- #1
3	Pptb- #2
4	Pptb- #3
5	Pptb- #4
6	Pptb- #5
7	Pptb- #16
8	Pptb- #17
9	Pptb- #18
10	Pptb- #19
11	Pptb- #20
12	Empty
13	Empty
14	Empty
15	EMpty
16	Empty
Row #2
Lane Number	Sample
1	Ladder (1kb plus)
2	Plep #1
3	Plep #2
4	Plep #3
5	Plep #4
6	Plep #5
7	Plep #16
8	Plep #17
9	Plep #18
10	Plep #19
11	Plep #20
12	Empty
13	Empty
14	Empty
15	EMpty
16	Empty

HTML tutorial

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09/1/13

3A Ligation

1) Clos Ori +Ptb- + pSB1A3

1) Clos Ori +Pveg1 + pSB1A3

1) Clos Ori +Pveg2 + pSB1A3

1) Clos Ori +Pliag + pSB1A3

1) mlSR + Plep + pSB1A3

1) mlSR +Terminator2 (4P) + pSB1A3

08/30/13

Gel of 3A Assembly

Row #1
Lane Number	Sample
1	2 #8
2	2 #10
3	2 #12
4	3 #15
5	3 #17
6	4 #1
7	4 #3
8	5 #1
9	6 #1
10	6 #3
11	Ladder (1kb plus)
12	Empty
13	Empty
14	Empty
15	EMpty
16	Empty

HTML tutorial

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08/30/13

Digest of 3A Assembly

3 #17

500ng X (μL/123.2ng) = 4.06μL

6 #1

500ng X (μL/98.7ng) = 5.07μL

6 #3

500ng X (μL/110.2ng) = 4.54μL

5 #1

500ng X (μL/162.6ng) = 3.08μL

2 #12

500ng X (μL/78.8ng) = 6.35μL

3 #15

500ng X (μL/131.8ng) = 3.79μL

4 #1

500ng X (μL/146.5ng) = 3.41μL

2 #8

500ng X (μL/117.7ng) = 4.25μL

2 #10

500ng X (μL/87.3ng) = 5.73μL

4 #3

500ng X (μL/82.9ng) = 6.03μL

Sample	EcoR1	Pst1	10x Buffer	DNA	PCR dH₂O	Total
3 #17	2μL	2μL	2μL	4.06μL	9.94μL	20μL
6 #1	2μL	2μL	2μL	5.07μL	8.93μL	20μL
6 #3	2μL	2μL	2μL	4.54μL	9.46μL	20μL
5 #1	2μL	2μL	2μL	3.08μL	10.92μL	20μL
2 #12	2μL	2μL	2μL	6.35μL	7.65μL	20μL
3 #15	2μL	2μL	2μL	3.79μL	10.21μL	20μL
4 #1	2μL	2μL	2μL	3.41μL	10.59μL	20μL
2 #8	2μL	2μL	2μL	4.25μL	9.75μL	20μL
2 #10	2μL	2μL	2μL	5.73μL	8.27μL	20μL
4 #3	2μL	2μL	2μL	6.03μL	7.97μL	20μL

Heat shock for 45 minutes in water bath and 15 minutes at 80°C on heat block to inactivate EcoR1

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08/30/13

Quantification and DNA Extraction of 3A

QIAprep Spin Miniprep Kit (250)

HTML tutorial

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08/27/13

Gel of 3A Assembly

Row #1
Lane Number	Sample
1	Ladder (1kb plus)
2	1 #9
3	2 #22
4	2 #24
5	2 #26
6	3 #3
7	4 #2
8	5 #3
9	5 #11
10	6 #2
11	6 #19
12	6 #24
13	Empty
14	Empty
15	EMpty
16	Empty

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08/26/13

Digest of Ter and Adh6

1 #9

500ng X (μL/124.7ng) = 4μL

ter #18

500ng X (μL/118.9ng) = 4.21μL

2 #22

500ng X (μL/76.1ng) = 6.57μL

2 #24

500ng X (μL/96.4ng) = 5.19μL

2 #26

500ng X (μL/99.4ng) = 5.03μL

3 #3

500ng X (μL/101ng) = 4.95μL

4 #2

500ng X (μL/104ng) = 4.81μL

5 #3

500ng X (μL/84.6ng) = 5.91μL

5 #11

500ng X (μL/124.6ng) = 4.01μL

6 #2

500ng X (μL/78.8ng) = 6.35μL

6 #19

500ng X (μL/141.4ng) = 3.54μL

6 #24

500ng X (μL/100.2ng) = 4.99μL

Sample	EcoR1	Pst1	10x Buffer	DNA	PCR dH₂O	Total
1 #9	2μL	2μL	2μL	4.21μL	9.79μL	20μL
2 #22	2μL	2μL	2μL	6.57μL	7.43μL	20μL
2 #24	2μL	2μL	2μL	5.19μL	8.81μL	20μL
2 #26	2μL	2μL	2μL	5.03μL	8.97μL	20μL
3 #3	2μL	2μL	2μL	4.95μL	9.05μL	20μL
4 #2	2μL	2μL	2μL	4.81μL	9.19μL	20μL
5 #3	2μL	2μL	2μL	5.91μL	8.09μL	20μL
5 #11	2μL	2μL	2μL	4.01μL	9.99μL	20μL
6 #2	2μL	2μL	2μL	6.35μL	7.65μL	20μL
6 #19	2μL	2μL	2μL	3.54μL	10.46μL	20μL
6 #24	2μL	2μL	2μL	4.99μL	9.01μL	20μL

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08/27/13

Quantification of 3A Assembly

QUIAPREP/Spin Miniprep Kit < (250)

HTML tutorial

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08/26/13

Gel of ter and Adh6

Row 1
Lane Number	Sample
1	Ladder (1kb plus)
2	ter #6
3	ter #18
4	ter #24
5	Adh6 #8
6	Adh6 #14
7	Adh6 #15
8	Empty

HTML tutorial

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08/26/13

Digest of Ter and Adh6

ter #6

500ng X (μL/124.7ng) = 4μL

ter #18

500ng X (μL/108ng) = 4.63μL

ter #24

500ng X (μL/315ng) = 1.59μL

Adh6 #8

500ng X (μL/300.3ng) = 12.41μL

Adh6 #14

500ng X (μL/95.4ng) = 5.24μL

Adh6 #15

500ng X (μL/328.3ng) = 1.52μL

Sample	EcoR1	Pst1	10x Buffer	DNA	PCR dH₂O	Total
ter #6	2μL	2μL	2μL	4.0μL	10.0μL	20μL
ter #18	2μL	2μL	2μL	4.63μL	9.37μL	20μL
ter #24	2μL	2μL	2μL	1.59μL	12.41μL	20μL
Adh6 #8	2μL	2μL	2μL	1.67μL	12.33μL	20μL
Adh6 #14	2μL	2μL	2μL	5.24μL	11.76μL	20μL
Adh6 #15	2μL	2μL	2μL	1.52μL	12.48μL	20μL

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08/26/13

DNA Extraction of Adh6 and Ter

QIAprep Spin Miniprep Kit (250)

HTML tutorial

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08/22/13

Transformation of 3A Assembly

Transform 4μL of ligation product into 100μL aliquots of competent E. coli cells
Incubate on ice for 30 minutes
Heat shock in water bath for 60 seconds
Incubate on ice for 5 minutes
Add 600μL of psi broth
Incubate at 37°C for 2 hours at 200 rpm
Make necessary dilutions and plate out

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08/22/13

3A Ligation

1) Clos Ori +Pveg2 (20G) + pSB1A3

1) Clos Ori +Plep (20E) + pSB1A3

1) Clos Ori +Pveg1 (23B) + pSB1A3

1) Clos Ori +Pliag (20A) + pSB1A3

1) mlSR +Terminator1 (6D) + pSB1A3

1) mlSR +Terminator2 (4P) + pSB1A3

Amount	Sample
2μL	Upstream Part
2μL	Downstream Part
2μL	Destination Backbone
2μL	10x T4 DNA Ligase Buffer
0.5μL	T4 DNA Ligase
10μL	PCR Water

Incubate at room temperature for 10 minutes, then at 80°C for 20 minutes to inactivate EcoR1

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08/15/13

Clos Ori

500ng X (440bp/2000bp) X (μL/190.5ng) = 0.577μL 1:105.7μL

mlSR

500ng X (750bp/2000bp) X (μL/260.4ng) = 0.908μL 1:109.08μL

Pliag

500ng X (121bp/2000bp) X (μL/150.1ng) = 0.201μL 1:102.01μL

Pveg1

500ng X (100bp/2000bp) X (μL/90.0ng) = 0.278μL 1:102.78μL

Plep

500ng X (157bp/2000bp) X (μL/235.0ng) = 0.167μL 1:101.67μL

Pveg2

500ng X (237bp/2000bp) X (μL/192.3ng) = 0.308μL 1:103.08μL

Terminator

500ng X (50bp/2000bp) X (μL/108.6ng) = 0.115μL 1:101.15μL

Terminator

500ng X (50bp/2000bp) X (μL/131.8ng) = 0.095μL 1:100.95μL

pSB1A3

500ng X (μL/31.8ng) = 2μL

Sample	EcoR1	Pst1	Xba1	Spe1	100x Bovine Serum Albumin	10x Buffer	DNA	PCR dH₂O	Total
Clos Ori	1μL	1μL	-	-	1μL	5μL	5.7μL	36.3μL	50μL
mlSR	1μL	1μL	-	-	1μL	5μL	9.08μL	32.92μL	50μL
Pliag	-	-	1μL	1μL	1μL	5μL	5.7μL	39.99μL	50μL
Pveg1	-	-	1μL	1μL	1μL	5μL	2.78μL	39.22μL	50μL
Pveg2	-	-	1μL	1μL	1μL	5μL	1.67μL	40.33μL	50μL
Plep	-	-	1μL	1μL	1μL	5μL	3.08μL	38.92μL	50μL
Terminator 6D	-	-	1μL	1μL	1μL	5μL	1.15μL	40.85μL	50μL
Temrinator 4P	-	-	1μL	1μL	1μL	5μL	0.95μL	41.05μL	50μL
pSB1A3	1μL	1μL	-	-	1μL	5μL	16μL	26μL	50μL

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08/21/13

PCR of BktB Fragments 1 & 2 used for SDM Step1

Previous attempt produced a lot of extraneous artifacts.

Primer concentration was too high and so was the template concentrations

Used Grad program previously used to run PCR machine

50°C to 60°V used 4 and 0

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08/21/13

Quantification of Backbones Post-SPRI Based Size Selection

HTML tutorial

The concentrations of pSB1A3 is low. Therefore, the SPRI based size selection was conducted on it once more. The gel results are displayed below.

HTML tutorial

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08/21/13

Transformation of Ter + pSB1C3 and Adh6 + pSB1C3

Thaw competent Top10 cell of E. coli from -80°C (100μL aliquots)
Add 20μL of ligation product to Top 10 E. coli
Incubate on ice for 30 minutes
Heat shock in water bath for exactly 60 seconds
Incubate on ice for 5 minutes
Add 600μL of psi broth
Incubate at 37°C for 2 hours at 200 rpm
Make dilutions using 90μL of psi broth: 1:10, 1:100, 1:1000
Plate out 25μL undiluted and diluted solutions onto a petri dish

Row 1
Lane Number	Sample
1	Ladder (1kb plus)
2	pSB1A3
3	Empty
4	Empty
5	Empty
6	Empty
7	Empty
8	Empty

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08/21/13

Gel Post SPRI Based Size Selection

Row 1
Lane Number	Sample
1	Ladder (1kb plus)
2	pSB1A3
3	pSB1C3
4	pSB1K3
5	Empty
6	Empty
7	Empty
8	Empty

HTML tutorial

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08/21/13

SPRI Based Size Selection

Use the instructions labeled “left side size slection.” This kit will hopefully remove the primer dimer from our product. Remove the primer dimer from the backbone PCR clean-up.

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08/21/13

Gel of Refined Backbone PCR

Row 1
Lane Number	Sample
1	Ladder (1kb plus)
2	pSB1A3
3	pSB1C3
4	pSB1K3
5	Empty
6	Empty
7	Empty
8	Empty

HTML tutorial

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08/21/13

PCR of pSB1A3, pSB1K3, pSB1K3 (refined)

Sample G in the primer matrix will be used as a template for this particular PCR

Label	Amount	Component
PCR # 1	3μL	SBprep-3P-1
	1μL	SBprep-2Ea
	2.0μL	pSB1A3
	94μL	PCR Supermix High Fidelity
PCR #3	3μL	SBprep-3P-1
	1μL	SBprep-2Ea
	2.0μL	pSB1C3
PCR #2	3μL	SBprep-3P-1
	1μL	SBprep-2Ea
	2.0μL	pSB1K3
	94μL	PCR Supermix High Fidelity

HTML tutorial

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08/20/13

Gel of Fifth PCR of Backbones

Row 1
Lane Number (1kb plus)	Sample
1	Ladder
2	E
3	G
4	Empty
5	Empty
6	Empty
7	Empty
8	Empty

HTML tutorial

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08/15/13

Use the two concentrations of E and G on 20-August

HTML tutorial

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08/20/13

PCR of Backbone Discussion

After discussing the results with Dr. Wawrik, it has been decided to cut the number of cycles down to thirty, and to increase the template used by two.

The best concentrations of primers to be used are indicated by the results of the primer matrix on letters E and G. F appeared to have been a probable pipetting error.

List of reactants and amount for primer matrix PCR optimization

Label	Amount	Component
E	0.5μL	SBprep-3P-1 (1:2 dilutions) use 1μL
	1.5μL	SBprep-2Ea
1.0μL	Template
	46.5μL	PCR Supermix High Fidelity
G	1.5μL	SBprep-3P-1
	0.5μL	SBprep-2Ea (1:2 dilutions) use 1μL
	1.0μL	Template
	46.5μL	PCR Supermix High Fidelity

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08/19/13

Gel of Ter and Adh6 Post Digest

Row 1
Lane Number (1kb plus)	Sample
1	Ladder
2	Adh6 #19
3	Adh6 #3
4	Ter #5
5	Ter #23
6	Ter #26
7	Ter #9
8	Empty

HTML tutorial

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08/14/13

Gel of pSB1K3, pSB1C3, pSB1A3 Stocks from 9-May

Row 1
Lane Number (1kb plus)	Sample
1	Ladder
2	pSB1A3
3	pSB1C3
4	pSB1K3
5	Empty
6	Empty
7	Empty
8	Empty

HTML tutorial

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08/14/13

PCR Clean-up Quanitification

HTML tutorial

Although the concentrations are good, the purity is still in question. It is an improvement on the PCR from 13-August

One possibility is that the original stocks used as templates in the PCR may not be pure enough. Therefore, a gel will be run on these original stocks

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08/14/13

Gel of pSB1K3, pSB1C3, pSB1A3 Post Clean-up

Row 1
Lane Number (1kb plus)	Sample
1	Ladder
2	pSB1A3
3	pSB1C3
4	pSB1K3
5	Empty
6	Empty
7	Empty
8	Empty

HTML tutorial

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08/14/13

PCR Clean-up of the Three Backbones

QIAquick PCR purification kit (250) is used before viewing the post clean-up product on a gel

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08/14/13

Gel of BktB Fragments

Row 1
Lane Number (1kb plus)	Sample
1	Ladder
2	50°C
3
4
5	60°C
6	Empty
7	Empty
8	Empty

What happened here? What is in the lanes above the comb?

Many have some product and we will use the forward and reverse primers to get the whole gene, if present

HTML tutorial

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08/14/13

Gel of pSB1K3, pSB1C3, pSB1A3 Pre Clean-up

Row 1
Lane Number (1kb plus)	Sample
1	Ladder
2	pSB1A3
3	pSB1C3
4	pSB1K3
5	Empty
6	Empty
7	Empty
8	Empty

HTML tutorial

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08/14/13

Quantified Backbone Stocks for PCR

The following stock are to be used for backbone amplification in the PCR procedure illustrated on 14-August

HTML tutorial

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08/14/13

Preparing an Equimolar solution of BktB Fragments for PCR to Stitch

Fragment 1: 340bp – 125.6ng/μL

Fragment 1: 660bp – 66.3ng/μL

Fragment 1: 300bp – 231.7ng/μL

Use 1μL of fragment 3 = 231.7ng/μL

Fragment 2:

(221.7) X (660/300) = 520ng x (1μL/66.3ng) = 7.7μL

Fragment 3:

(221.7) X (340/300) = 263ng x (1μL/125.6ng) = 2.1μL

Added to a 1.5mL epi tube and diluted to 200μL

1004ng of DNA/ 200μL = 5ng/μL final concentration of DNA

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08/14/13

PCR of i-GEM Plasmid Backbones Continued

PCR #1
Amount (μL)	Component
100	PCR Supermix High Fidelity
0.7	SB-prep-3P-1
0.7	SB-prep-2Ea
0.6	pSB1A3 (1ng/μL)
PCR #2
Amount (μL)	Component
100	PCR Supermix High Fidelity
0.7	SB-prep-3P-1
0.7	SB-prep-2Ea
0.6	pSB1C3 (1ng/μL)
PCR #3
Amount (μL)	Component
100	PCR Supermix High Fidelity
0.7	SB-prep-3P-1
0.7	SB-prep-2Ea
0.6	pSB1K3 (1ng/μL)

HTML tutorial

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08/14/13

Adjusted PCR of iGEM Plasmid Backbones

Diluting concentrations of pSB1A3, pSB1C3, pSB1K3 to 1ng/μL

Quantification of current backbones to confirm concentrations before conducting dilution

HTML tutorial

pSB1A3

Vf = (27.5ng/μL) X (1μL) X (1μL/1ng) = 27.5μL – 1μL of template = 26.5μL of PCR water

pSB1C3

Vf = (39.9ng/μL) X (1μL) X (1μL/1ng) = 39.9μL – 1μL of template = 38.9μL of PCR water

pSB1K3

Vf = (29.4ng/μL) X (1μL) X (1μL/1ng) = 29.4μL – 1μL of template = 28.4μL of PCR water

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08/13/13

Quantification of PCR Clean-up

The concentrations are good, however the purity of the product is not. Actions taken to remedy this problem are to run another PCR using 1ng/μL of template and running the third step of the PCR cycle at 68°C for 5 minutes.

HTML tutorial

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08/13/13

Gel of pSB1K3, pSB1C3, pSB1A3 Post Clean-up

Row 1
Lane Number	Sample
1	Ladder
2	pSB1A3
3	pSB1C3
4	pSB1K3
5	Empty
6	Empty
7	Empty
8	Empty

HTML tutorial

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08/13/13

PCR Clean-up of pSB1K3, pSB1C3, pSB1A3

The three backbones were cleaned up using QIAquick PCR Purification Kit (250) so that the next gel will much more clearly display the backbone.

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08/13/13

Gel of pSB1K3, pSB1C3, pSB1A3

Row 1
Lane Number	Sample
1	Ladder
2	pSB1K3
3	pSB1C3
4	pSB1A3
5	Empty
6	Empty
7	Empty
8	Empty

HTML tutorial

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08/13/13

Plasmid Extraction of AaDc-, AaDc+, Thl+, Thl-, and Ptb+

QUIAPREP/Spin Miniprep Kit < (250)

Cells were centrifuged from an inoculated broth

HTML tutorial

None of the concentrations obtained are high enough to work with. Must go about finding a more efficient plasmid extraction for these samples.

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08/13/13

PCR of i-GEM Plasmid Backbones

PCR #1
Amount (μL)	Component
100	PCR Supermix High Fidelity
0.7	SB-prep-3P-1
0.7	SB-prep-2Ea
0.6	pSB1C3 (43ng/μL)
PCR #2
Amount (μL)	Component
100	PCR Supermix High Fidelity
0.7	SB-prep-3P-1
0.7	SB-prep-2Ea
0.6	pSB1C3 (74.8ng/μL)
PCR #3
Amount (μL)	Component
100	PCR Supermix High Fidelity
0.7	SB-prep-3P-1
0.7	SB-prep-2Ea
0.6	pSB1C3 (55.1ng/μL)

HTML tutorial

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08/14/13

Post-Cleanup of BktB PCR Products

All Fragment 1’s, lanes 8 and 9 of Fragment 2, and all Fragment

HTML tutorial

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08/13/13

PCR of BktB from PCR Tempalte

HTML tutorial

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08/9/13

Gel of BktB Fragments

Row #1
Lane Number	Sample	Temperature (°C)
1	Ladder
2	Fragment 1	50
3	Fragment 1
4	Fragment 1
5	Fragment 1	60
6	Fragment 2	50
7	Fragment 2
8	Fragment 2
9	Fragment 2	60
10	Fragment 3	50
11	Fragment 3
12	Fragment 3
13	Fragment 3	60
14	Empty
15	EMpty
16	Empty

HTML tutorial

Based on 2% agarose gel, 115V for about 30 minutes

Going to PCR the whole gene to see if I can reduce non-specific products in fragment #2

HTML tutorial

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08/8/13

PCR of BktB Fragments

Preformed gradient PCR for all 3 BktB fragments

Undiluted template was used

HTML tutorial

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08/7/13

Transformation of Top10 with ter in pSB1C3

Thaw competent 100μL aliquot of E. coli cells from -80°C freezer
Add 20μL of ligation poduct to Top10 E. coli
Incubate on ice for 30 minutes
Heat shock in water bath for 60 seconds
Incubate on ice for 5 minutes
Add 600μL of psi broth
Incubate at 37°C for 2 hours at 200 rpm
Make a dilution series of 1:10, 1:100, 1:1000, and 1:10000

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08/7/13

Ligation of pSB1C3 and Adh6

Into 1.5mL microcentrifuge tubes

Amount (μL)	Component
20	Heat killed digest of pSB1C3
20	Heat killed digest of Adh6
1.0	T4 Ligase
5.0	10X T4 Ligase Buffer
4.0	Nuclease Free PCR Water
50	Total

Incubate on bench for 45 minutes

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08/9/13

digestion of ter

Sample	EcoR1	Pst1	10x Buffer	DNA	PCR dH₂O	Total
ter #23	1μL	1μL	1μL	2.5μL	14.5μL	20μL
ter #7	1μL	1μL	1μL	1.5μL	15.5μL	20μL
ter #26	1μL	1μL	1μL	2μL	15μL	20μL
ter #5	1μL	1μL	1μL	2μL	15μL	20μL
pSB1C3	1μL	1μL	1μL	2.5μL	14.5μL	20μL

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08/9/13

Plasmid Extraction of AaDc- and Ptb-

HTML tutorial

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08/7/13

Restriction Digest of Ccell Adh1 and Adh2

Adh1 #5

500ng X (μL/297.2ng) = 1.68μL

Adh1 #19

500ng X (μL/89.3ng) = 5.60μL

Adh1 #24

500ng X (μL/261.6ng) = 1.91μL

Adh1 #4

500ng X (μL/264.0ng) = 1.89μL

Adh1 #13

500ng X (μL/588.6ng) = 0.85μL

Sample	EcoR1	Pst1	10x Buffer	DNA	PCR dH₂O	Total
Adh1 #5	2μL	2μL	2μL	1.5μL	12.5μL	20μL
Adh1 #19	2μL	2μL	2μL	5.5μL	8.5μL	20μL
Adh1 #24	2μL	2μL	2μL	2μL	12μL	20μL
Adh1 #4	2μL	2μL	2μL	2μL	12μL	20μL
Adh1 #13	2μL	2μL	2μL	1μL	13μL	20μL

Sample	EcoR1	Pst1	10x Buffer	DNA	PCR dH₂O	Total
Adh6 #5	1μL	1μL	1μL	5.0μL	12.0μL	20μL
Adh6 #19	1μL	1μL	1μL	2.0μL	15.0μL	20μL
Adh6 #3	1μL	1μL	1μL	3.0μL	14.0μL	20μL
Adh6 #15	1μL	1μL	1μL	3.0μL	14.0μL	20μL
pSB1C3	1μL	1μL	1μL	2.5μL	14.5μL	20μL

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08/7/13

Gel for Post-Digest of Promoters and Terminators

Using 2% agarose in the gel

Gel 1
Lane Number	Sample
1	Ladder
2	#16-6D
3	#16-23B
4	#9-4P
5	#9-6D
6	#3-23B
7	#14-20E
8	Empty

HTML tutorial

Gel 2
Lane Number	Sample
1	Ladder
2	#15-20A
3	#7-20G
4	#7-4D
5	#14-20A
6	#10-4P
7	#11-20E
8	Empty

HTML tutorial

Samples display a possible insert, and will be sent off for sequencing

3-23B, 16-23B, 14-20E, 15-20A, 16-6D, 14-20A, and 10-4P

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08/7/13

Modified Restriction Digest of Promoters and Terminators

HTML tutorial

Sample	EcoR1	Pst1	10x Buffer	DNA	PCR dH₂O	Total
#16-6D	1μL	1μL	1μL	10.0μL	7.0μL	20μL
#16-23B	1μL	1μL	1μL	10.0μL	7.0μL	20μL
#9-4P	1μL	1μL	1μL	10.0μL	7.0μL	20μL
#9-6D	1μL	1μL	1μL	10.0μL	7.0μL	20μL
#3-23B	1μL	1μL	1μL	10.0μL	7.0μL	20μL
#14-20E	1μL	1μL	1μL	10.0μL	7.0μL	20μL
#15-20A	1μL	1μL	1μL	10.0μL	7.0μL	20μL
#7-20G	1μL	1μL	1μL	10.0μL	7.0μL	20μL
#7-4D	1μL	1μL	1μL	10.0μL	7.0μL	20μL
#14-20A	1μL	1μL	1μL	10.0μL	7.0μL	20μL
#10-4P	1μL	1μL	1μL	10.0μL	7.0μL	20μL
#11-20E	1μL	1μL	1μL	10.0μL	7.0μL	20μL

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08/7/13

Plasmid Extraction of Ccell of Adh1 and Adh2

QIAPrep Spin Miniprep Kit (250)

The gene’s origin is unknown, and will be found upon sequencing

HTML tutorial

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07/18/13

Plasmid Prep of Transformed Top10 Cells with pSB1C3 + mlsR and puc19 + KIVD

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07/17/13

DNA Extraction from Ca, Cs, Cb, and Cc using MoBio Power Soil DNA Extraction Kit

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07/11/13

Dual Digest of pSB1C3 and mlsR Part

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07/25/13

Gel of Aadc-/+, thl+/-, Ptb-/+ from 1 Aug

Row #1
Lane Number	Sample
1	Ladder
2	Aadc- #6
3	Aadc- #23
4	Aadc+ #20
5	thl- #4
6	Aadc+ #15
7	Aadc+ #2
8	thl+ #17
9	thl+ #3
10	thl+ #10
11	thl- #14
12	thl- #21
13	Empty
14	Empty
15	EMpty
16	Empty
Row #2
Lane Number	Sample
1	Ladder
2	Empty
3	Empty
4	Empty
5	Empty
6	Empty
7	Ladder
8	Ptb- #5
9	Ptb+ #9
10	Ptb+ #1
11	Ptb+ #26
12	Ptb- #18
13	Ptb- #8
14	Empty
15	Empty
16	Empty

HTML tutorial

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08/02/13

Plasmid Extraction of Ccell Adh and Csac Adh

QIAPrep Spin Miniprep Kit (250)

HTML tutorial

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08/02/13

Restriction Digest of Aadc-/+ and Ptb-/+

#2 Aadc+ 500ng (^1&#956L⁄_85.9ng) = 6.0μL

#15 Aadc+ 500ng (^1&#956L⁄_117.5ng) = 4.5μL

#20 Aadc+ 500ng (^1&#956L⁄_92.1ng) = 5.5μL

#23 Aadc- 500ng (^1&#956L⁄_184.8ng) = 3.0μL

#6 Aadc- 500ng (^1&#956L⁄_218.1ng) = 2.5μL

#17 thl+ 500ng (^1&#956L⁄_100.0ng) = 5.0μL

#3 thl+ 500ng (^1&#956L⁄_84.4ng) = 6.0μL

#10 thl+ 500ng (^1&#956L⁄_90.4ng) = 5.5μL

#14 thl- 500ng (^1&#956L⁄_85.7ng) = 6.0μL

#21 thl- 500ng (^1&#956L⁄_89.1ng) = 5.5μL

#4 thl- 500ng (^1&#956L⁄_131.8ng) = 4.0μL

#9 Ptb+ 500ng (^1&#956L⁄_81.4ng) = 6.0μL

#1 Ptb+ 500ng (^1&#956L⁄_124.2ng) = 4.0μL

#26 Ptb+ 500ng (^1&#956L⁄_206.7ng) = 2.5μL

#18 Ptb- 500ng (^1&#956L⁄_103.7ng) = 5.0μL

#8 Ptb- 500ng (^1&#956L⁄_150.1ng) = 3.5μL

#5 Ptb- 500ng (^1&#956L⁄_216.0ng) = 2.5μL

Sample	EcoR1	Pst1	10x Buffer	DNA	PCR dH₂O	Total
#2 Aadc+	2μL	2μL	2μL	6.0μL	8.0μL	20μL
#15 Aadc+	2μL	2μL	2μL	4.5μL	9.5μL	20μL
#20 Aadc+	2μL	2μL	2μL	5.5μL	8.5μL	20μL
#23 Aadc-	2μL	2μL	2μL	3.0μL	11.0μL	20μL
#6 Aadc-	2μL	2μL	2μL	2.5μL	11.5μL	20μL
#17 thl+	2μL	2μL	2μL	5.0μL	9.0μL	20μL
#3 thl+	2μL	2μL	2μL	6.0μL	8.0μL	20μL
#10 thl+	2μL	2μL	2μL	5.5μL	8.5μL	20μL
#14 thl-	2μL	2μL	2μL	6.0μL	8.0μL	20μL
#21 thl-	2μL	2μL	2μL	5.5μL	8.5μL	20μL
#4 thl-	2μL	2μL	2μL	4.0μL	10.0μL	20μL
#9 Ptb+	2μL	2μL	2μL	6.0μL	8.0μL	20μL
#1 Ptb+	2μL	2μL	2μL	4.0μL	10.0μL	20μL
#26 Ptb+	2μL	2μL	2μL	2.5μL	11.5μL	20μL
#18 Ptb-	2μL	2μL	2μL	5.0μL	9.0μL	20μL
#8 Ptb-	2μL	2μL	2μL	3.5μL	10.5μL	20μL
#5 Ptb-	2μL	2μL	2μL	2.5μL	11.5μL	20μL

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08/02/13

Plasmid Extraction of Aadc-/+, Ptb-/+, thl-/+

QIAPrep Miniprep Kit (250)

HTML tutorial

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08/01/13

Gel of Regulatory Promoters and Terminators (1 Aug)

Row 1
Lane Number	Sample
1	Ladder
2	3:23B
3	10:4P
4	12:20G
5	9:6D
6	16:6D
7	8:20G
8	11:20E
9	14:20E
10	15:20A
11	14:20A
12	9:4P
13	16:23B
14	9:20G
15	7:20G

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08/1/13

Restriction Digest using EcoR1 and Pst1 (26 July Quantification)

#16-6D 500ng (^1&#956L⁄_108.6ng) = 4.5μL

#14-20E 500ng (^1&#956L⁄_235.2ng) = 2.0μL

#12-20G 500ng (^1&#956L⁄_135.1ng) = 3.5μL

#8-20G 500ng (^1&#956L⁄_192.3ng) = 2.5μL

#14-20A 500ng (^1&#956L⁄_156.5ng) = 3.0μL

#9-6D 500ng (^1&#956L⁄_`99.7ng) = 2.5μL

#10-4P 500ng (^1&#956L⁄_131.8ng) = 4.0μL

#7-20G 500ng (^1&#956L⁄_232.8ng) = 2.0μL

#3-23B 500ng (^1&#956L⁄_90.0ng) = 5.5μL

#16-23B 500ng (^1&#956L⁄_123.5ng) = 4.0μL

#9-4P 500ng (^1&#956L⁄_145.2ng) = 3.5μL

#9-20G 500ng (^1&#956L⁄_106.8ng) = 4.5μL

#15-20A 500ng (^1&#956L⁄_150.1ng) = 3.5μL

#11-20E 500ng (^1&#956L⁄_126.2ng) = 4.0μL

Sample	EcoR1	Pst1	10x Buffer	DNA	PCR dH₂O	Total
#16-6D	2μL	2μL	2μL	4.5μL	9.5μL	20μL
#14-20E	2μL	2μL	2μL	2.0μL	12.0μL	20μL
#12-20G	2μL	2μL	2μL	3.5μL	10.5μL	20μL
#8-20G	2μL	2μL	2μL	2.5μL	11.5μL	20μL
#14-20A	2μL	2μL	2μL	3.0μL	11.0μL	20μL
#9-6D	2μL	2μL	2μL	2.5μL	11.5μL	20μL
#10-4P	2μL	2μL	2μL	4.0μL	10.0μL	20μL
#7-20G	2μL	2μL	2μL	2.0μL	12.0μL	20μL
#3-23B	2μL	2μL	2μL	5.5μL	8.5μL	20μL
#16-23B	2μL	2μL	2μL	4.0μL	10.0μL	20μL
#9-4P	2μL	2μL	2μL	3.5μL	10.5μL	20μL
#9-20G	2μL	2μL	2μL	4.5μL	9.5μL	20μL
#15-20A	2μL	2μL	2μL	3.5μL	10.5μL	20μL
#11-20E	2μL	2μL	2μL	4.0μL	10.0μL	20μL

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08/1/13

Plasmid Extraction of Terminators and Promoters

QIAPrep Spin Miniprep Kit (250)

HTML tutorial

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08/01/13

Ampicillin 1x Preparation

To make 40mL of ampicillin 1x antibiotic

2.0g ampicillin 1000x stock from 4°C refrigerator
Pour the amipicillin ino a 50mL falcon
Fill flacon wih dH₂O to 40mL mark
Use 10mL syringe with puradisc 25mm filter to dispense 10mL of ampicillin 1x into four 10mL falcons (leaves alternative stocks if one is contaminated)

Ampicillin 1000x = 50ng/mL

50ng/mL x 40mL = 2.0g Ampicillin 1000x

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07/31/13

Promoter and Terminator Culture Preparation

Used 2XYT liquid medium instead of LB to increase carbon amount in order to enhance growth of cultures.

-3mL of 2XYT with Chloramphenicol antibiotic

-Pellet of culture

Incubated at 37°C with shaking at 220rpm

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07/31/13

2XYT Medium Preparation

(Agar: 10g)
Bacto Tryptone: 16g
Yeast Extract: 10g
Sodium Chloride: 5g
Distilled Water: 1000mL

Fill 1L jar with 500mL of dH₂O. Pour components and magnetic stirring rod into jar. Fill jar with another 500mL of dH₂O until the shoulder of the jar is reached.

Autoclave for 20 minutes at slow exhaust (Setting #2)

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07/31/13

Gel of DNA Preps from R. eutropha

Run a gel with 3μL of dye and 5μL of DNA extract for each of our R. eutropha extracts.

Row #1
Lane Number	Sample
1	Ladder
2	Phenol Chloroform (27 July)
3	R. eutropha 1 (24 July)
4	R. eutropha 2 (24 July)
5	Power Soil (25 July)
6	Phenol Chloroform (25 July)
7	Empty
8	Empty

NOTE: Lane six shows chewed up DNA by nucleases

Disposed all samples except for Power Soil because it has the highest efficacy

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07/30/13

Plasmid Purification

Plasmid purification of top 10 cells transformed with ligation of pSB1C3 and Ca promoters (Ptb-RBS, Ptb+RBS, thl+RBS, thl-RBS, Aadc+RBS, AaDc-RBS)

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07/30/13

Plasmid Extraction of Terminators and Promoters

Used QIAPrep Spin Miniprep Kit (250)

HTML tutorial

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07/27/13

Dual Re gel of Regulatory Terminators and Promoters

Row 1
Lane Number	Sample
1	Ladder
2	#14-20A
3	#17-20G
4	#14-20E
5	#11-20E

HTML tutorial

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07/27/13

Dual Digest of pSB1C3 and mlsR Part

Row 1
Lane Number	Sample
1	Ladder
2	#10-4p
3	#14-20E
4	#14-20A
5	#7-20G
6	#11-20E
7	#16-20E
8	#9-4P
9	#15-20A

HTML tutorial

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07/11/13

Dual Digest of pSB1C3 and mlsR Part

500ng in 20μL with 2μL for each digest

#11-20E 500ng (^1&#956L⁄_58.1ng) = 8.5μL

#9-4P 500ng (^1&#956L⁄_40.2ng) = 12.5μL

#14-20A 500ng (^1&#956L⁄_43.5ng) = 12.5μL

#7-20G 500ng (^1&#956L⁄_59.4ng) = 12.5μL

#14-20E 500ng (^1&#956L⁄_37.5ng) = 12.5μL

#10-4P 500ng (^1&#956L⁄_43.6ng) = 12.5μL

#15-20A 500ng (^1&#956L⁄_51.6ng) = 12.5μL

#16-23B 500ng (^1&#956L⁄_52.5ng) = 12.5μL

Sample	EcoR1	Pst1	10x Buffer	DNA	PCR dH₂O	Total
#11-20E	2μL	2μL	2μL	8.5μL	5.5μL	20μL
#9-4P	2μL	2μL	2μL	12.5μL	1.5μL	20μL
#14-20A	2μL	2μL	2μL	11.5μL	2.5μL	20μL
#7-20G	2μL	2μL	2μL	8.5μL	5.5μL	20μL
#14-20E	2μL	2μL	2μL	13.5μL	0.5μL	20μL
#10-4P	2μL	2μL	2μL	11.5μL	2.5μL	20μL
#15-20A	2μL	2μL	2μL	9.5μL	4.5μL	20μL
#16-23B	2μL	2μL	2μL	9.5μL	4.5μL	20μL

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07/26/13

Nano Drop of Isolated DNA from July 25

HTML tutorial

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07/26/13

Gel of BktB piece 3 PCR using DNA Extracts

Row #1
Lane Number	Sample
1	Ladder
2	R. eutropha extract (27 July)
3	R. eutropha 1 (24 July)
4	R. eutropha 2 (24 July)
5	Power Soil (25 July)
6	Phenol Chloroform (25 July)
7	Empty
8	Empty

HTML tutorial

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07/26/13

Gel Repeat of BktB PCR Gradient (25 July)

Repeated gel with larger loading of DNA. Removing only loaded 5μL of dye pls products. Wanted to see if bands appear brighter.

Row #1
Lane Number	Sample	Temperature (°c)
1	Kb Plus Ladder
2	Piece 1	50
3	Piece 1	52.7
4	Piece 1	54.5
5	Piece 1	56.4
6	Piece 1	60
7	Piece 2	50
8	Piece 2	52.7
9	Piece 2	54.5
10	Piece 2	56.4
11	Piece 2	60
12	Ladder
13	Empty
14	Empty
15	EMpty
16	Empty

HTML tutorial

Row #1
Lane Number	Sample	Temperature (°c)
1	Kb Plus Ladder
2	Piece 3	50
3	Piece 3	52.7
4	Piece 3	54.5
5	Piece 3	56.4
6	Piece 3	60
7	Whole gene	50
8	Whole gene	52.7
9	Whole gene	54.5
10	Whole gene	56.4
11	Whole gene	60
12	Ladder
13	Empty
14	Empty
15	Empty
16	Empty

HTML tutorial

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07/11/13

PCR of BktB piece 3 using Different DNA Extraction

Wanted to identify which extracts may have DNA present

Previous gradient seems to have produced a PCR product for 3 piece. Used temperature 55°C, since this is the predicted optimal temperature for the SDM2 forward and the BktB_Rev primers

Templates used:

R. eutropha = 27 July_ 16.9
R. eutropha = 24 July_ 25
R. eutropha = 24 July_ 38.9
Power Soil Extraction = 25 July_ 28.3
Phenol Chloroform Extraction = 25 July_ 637

HTML tutorial

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07/25/13

Gel of Gradient PCR for BktB Site-Directed Mutagenesis

Row #1
Lane Number	Sample	Temperature (°c)
1	Kb Plus Ladder
2	Forward + SDM1 Reverse	50
3	Forward + SDM1 Reverse	52.7
4	Forward + SDM1 Reverse	54.5
5	Forward + SDM1 Reverse	56.4
6	Forward + SDM1 Reverse	60
7	SDM1 Forward + SDM2 Reverse	50
8	SDM1 Forward + SDM2 Forward	52.7
9	SDM1 Forward + SDM2 Reverse	54.5
10	SDM1 Forward + SDM2 Reverse	56.4
11	SDM2 Forward + Reverse	60
12	Empty
13	Empty
14	Empty
15	EMpty
16	Empty
Row #2
Lane Number	Sample	Temperature (°C)
1	Ladder
2	SDM2 Forward + Reverse	50
3	SDM2 Forward + Reverse	52.7
4	SDM2 Forward + Reverse	54.5
5	SDM2 Forward + Reverse	56.4
6	SDM2 Forward + Reverse	60
7	Forward + Reverse	50
8	Forward + Forward	52.7
9	Forward + Reverse	54.5
10	Forward + Reverse	56.4
11	Forward + Reverse	60
12	Empty
13	Empty
14	Empty
15	Empty
16	Empty

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07/25/13

DNA Extraction from R. eutropha

If DNA template is the problem with the PCR, then one of the following procedures may correct it.

1) Phenol Chloroform extraction using the protocol from July 24, 2013 with the following modification: 2μL of lysozyme added in the 3rd step with the 10% SDS, to encourage membrane disolution

2)Using the MoBio Power Soil DNA extraction kit and included protocol.

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07/25/13

Transformation of Ca Promoters in pSB1C3

Most plates had growth. Plates are going to be incubated at 37°c for another night. Tomorrow a library will be made.

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07/26/13

Transformation of Ca Promoters in pSB1C3

Colonies were larger. Transformant libraries were made of Ca promoters in addition to ter, Adh6, and C. cell Adh. Plates were parafilmed and placed in 37°C incubator to prevent drying out over the weekend. Monday, plates will be streaked, Tuesday, plasmids will be extracted, and Wednesday, plasmids will be sent for sequencing.

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07/25/13

PCR of BktB for Site-Directed Mutagenesis

Repeated PCR of BktB using primers for sie-directed mutagenesis

Also included one PCR series using the Forward and Revers primers for the whole gene

Used only undiluted templates

Temperature gradient was selected to identify correct temperature for each pair of primers (assuming template is present)

Used wells:

1: T = 50&#176:c

4: T = 52.7&#176:c

6: T = 54.5&#176:c

8: T = 56.4&#176:c

12: T = 60&#176:c

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07/25/13

Gel of BktB Site-Directed Mutagenesis PCR

Row #1
Lane Number	Sample	Dilution
1	Empty
2	Kb plus ladder
3	Forward + SDM1 Reverse	1:10
4	Forward + SDM1 Reverse	1:100
5	Forward + SDM1 Reverse	1:1000
6	Forward + SDM1 Reverse	1:10000
7	SDM1 Forward + SDM2 Reverse	1:10
8	SDM1 Forward + SDM2 Forward	1:100
9	SDM1 Forward + SDM2 Reverse	1:1000
10	SDM1 Forward + SDM2 Reverse	1:10000
11	SDM2 Forward + Reverse	1:10
12	SDM2 Forward + Reverse	1:100
13	SDM2 Forward + Reverse	1:1000
14	SDM@ Forward + Reverse	1:10000
15	Kb plus Ladder
16	Empty

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No PCR products.

Will run a temperature gradient for each sample to see if temperature is affecting enzyme activity

Will preform Power Soil DNA Extraction and bead beaten with phenol: chloroform extraction and isopropanol precipitation to see if template is the problem.

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07/24/13

Site-Directed Mutagenesis PCR for BktB

Into each tube:

Mix: 98μL
Template: 2μL
Total: 100μL

Dilutions used: 1:10, 1:100, 1:1000, 1:10000

Mix:

2x Master Mix: 250μL
Forward Primer: 3μL
Reverse Primer: 3μL
PCR dH₂O: 244μL
Total: 500μL

PCR Primers Used:

Forward + SDM1 Reverse

340 base pairs

SDM1 Forward + SDM2 Revers

660 base pairs

SDM2 Forward + Reverse

300 base pairs

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07/24/13

DNA Extraction of R. eutropha

Re suspend cells in 500μL 1x STE buffer
Vortex until mixture is homogenous
Add 50μL of 10% Sodium-dodecyl sulfate (SDS)
IF cells insufficiently lysed, 2 minutes in bead beater
Add 500μL of Chloroform:Phenol pH 8

Extract bottom layer (2 phases present)
Minimize time the chloroform is out of fridge

Vortex
Centrifuge for 30 seconds

14000 rpm

Transfer top phase to 1.5mL microcentrifuge tube

Don't take up white precipitant from interphase of solution

Add 500μL of isoamyl acetate (CIAA 24:1) to top phase
Vortex
Centrifuge for 30 seconds

14000 rpm

Transfer top phase to 1.5mL microcentrifuge tube

Don't take up white precipitant from interphase of solution

REPEAT STEPS 9-12

Note: Keep track of amount finally transfered in step 12

Add (2/10) volume (amount of top phase) of 3M Sodium Acetate
Add (7/10) volume (step 14 amount) of Isopropanol

Note: Mix solutions with pipette

Centrifuge for 25 minutes

14000 rpm

Dispose of liquid portion

Don't disturb invisible pellet

Dry pellet on heat block at 60°C for 10 minutes

Leave caps open for evaporation of isopropanol

Re suspend pellet in 50μL of distilled water

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07/24/13

Repeat of Transformation of Ca Promoters into Top 10 Cells

Preformed as outlined on February 11, 2013

-Last time we used 2μL for transformation this time since our ligations will contain a maximum of 100ng of insert (recommended amount for transformation of top 10 cells), we are going to use the entire ligation volume (20μL)

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07/23/13

Making Beef Extract Medium

Component	Amount
Beef Extract	3g
Peptone	5g
Agar	15g
Distilled Water	1000mL

Pour components and one magnetic mixer into empty jar. Fill jar with distilled water to 1000mL mark.

Autoclave at setting number "2" for 20 minutes at slow exhaust

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07/23/13

PCR of Ca AdhE

Component	Amount
2X Master Mix	125μL
Forward Primer	4μL
Reverse Primer	4μL
PCR Water	117μL
TOTAL	250μL

1 Reaction 50μL = 49μL Mix + 1μL Template

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07/23/13

Digest of Cc Adh and Cs Adh

500ng DNA for each reaction

500ng (^1&#956L⁄_39.7ng) = 12.5 μL Cc DNA

500ng (^1&#956L⁄_36.0ng) = 14 μL Cs DNA

Component	Cc	Cs	pSB1C3
10X Buffer	2μL	2μL	2μL
EcoR1	2μL	2μL	2μL
Pst1	2μL	2μL	2μL
DNA	12.5μL	14μL	2.5μL
PCR Water	1.5μL	0μL	11.5μL
Total	20μL	20μL	20μL

500ng (^1&#956L⁄_202ng) = 2.5μL pSB1C3 backbone

Incubate at 37°C water bath for 45 minutes

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07/11/13

Repeat of Cohesive End CLoning of gBloeu Ca Promoters Ptb, AaDc, and thl into pSB1C3 Vector

1. Digest

A. In 1 tube contain

Backbone: 2μL
EcoR1: 1μL
Pst1: 1μL
10x Buffer: 2μL
Water: 14μL
Total: 20μL

B. In each 6 tubes combine

gBloeu Resuspension: 10μL
EcoR1: 1μL
Pst1: 1μL
10x Buffer: 2μL
Water: 6μL
Total: 20μL

Incubate in 37°C water bath for 1 hour

Clean with QIAquick PCR Purification Kit

Nano drop

Aadc quantification appears to be good with a reading of 1.9ng/μL, but is the lowest limit of detection for the nano drop 1000, so we can't be sure

Ligation

Since the vector is 31.7 ng/μL and the protocol calls for 50ng of DNA and the concentration of insert might be 1.5 to 2.0ng/μL due to the need of 20ng.

pSB1C3: 2μL
Promoter: 13μL
Buffer: 2μL
Ligase: 1μL
Water: 2μL
Total: 20μL

Incubate at room temperature overnight

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07/23/13

Gel of AdhE and AdhE1

Row #1
Lane Number	Sample	Dilution	Temperature(μ)
1	Ladder
2	Ca AdhE	1:100	53
3	Ca AdhE	1:100	50
4	Ca AdhE	1:100	56
5	Ca AdhE	1:100	60
6	Ca AdhE	1:10	50
7	Ca AdhE	1:10	53
8	Ca AdhE	1:10	56
9	Ca AdhE	1:10	60
10	Ca AdhE	1:1000	50
11	Ca AdhE	1:1000	53
12	Ca AdhE	1:1000	56
13	Ca AdhE	1:1000	60
14	Empty
15	Empty
16	Empty
Row #2
1	Ladder
2	Ca AdhE1	1:100	50
3	Ca AdhE1	1:10	50
4	Ca AdhE1	1:100	53
5	Ca AdhE1	1:100	50
6	Ca AdhE1	1:100	56
7	Ca AdhE1	1:10	53
8	Ca AdhE1	1:10	56
9	Ca AdhE1	1:10	60
10	Ca AdhE1	1:1000	50
11	Ca AdhE1	1:1000	53
12	Ca AdhE1	1:1000	56
13	Ca AdhE1	1:1000	60
14	Empty
15	Empty
16	Empty

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07/22/13

PCR of Ca AdhE and AdhE1

Sample	Dilution	50°	53°	56°	60°
Empty
Ca AdhE	1:10	✓	✓	✓	✓
Ca AdhE	1:100	✓	✓	✓	✓
Ca AdhE	1:1000	✓	✓	✓	✓
Empty
Ca AdhE1	1:10	✓	✓	✓	✓
Ca AdhE1	1:100	✓	✓	✓	✓
Ca AdhE1	1:1000	✓	✓	✓	✓

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07/22/13

PCR of Ca AdhE/AdhE1 using pSOL

Used Qia Quick Prep Plasmid Extraction Kit

AdhE and AdhE1 are supposed to be located on pSOL plasmid using pSOL as template (1:10, 1:100, 1:1000)

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07/22/13

Plasmid Extraction of Ca pSOL

Used Qia Quick Prep Plasmid Extraction Kit

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07/11/13

Dual Gel of mlsR Xformants

Row #1
Well #	Sample
1	Ladder
2	mlsR 5
3	mlsR 9
4	mlsR 10
5	mlsR 12
6	mlsR 22
7	mlsR 24

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07/22/13

Dual Digest of mlsR Xformants

EcoR1 and Pst1 Dual Digest of mlsR colonies

5, 9, 10, 12, 22, 24

500ng (^1&#956L⁄_112.2ng) = 4.45 μL → 4.5 μL

500ng (^1&#956L⁄_124.5ng) = 4.02 μL → 4.0 μL

500ng (^1&#956L⁄_117.7ng) = 4.25 μL → 4.5 μL

500ng (^1&#956L⁄_162.0ng) = 3.09 μL → 3.0 μL

500ng (^1&#956L⁄_188.7ng) = 2.65 μL → 2.5 μL

500ng (^1&#956L⁄_206.4ng) = 2.42 μL → 2.5 μL

Colony Number	EcoR1	Pst1	10x Buffer	DNA	PCR dH₂O	Total
5	2μL	2μL	2μL	4.5μL	9.5μL	20μL
9	2μL	2μL	2μL	4.0μL	10.0μL	20μL
10	2μL	2μL	2μL	4.5μL	9.5μL	20μL
12	2μL	2μL	2μL	3.0μL	11.0μL	20μL
22	2μL	2μL	2μL	2.5μL	11.5μL	20μL
24	2μL	2μL	2μL	2.5μL	11.5μL	20μL

Incubated at 37°L for 45 minutes

Transformed to 80°L heat block for 1 hour

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07/22/13

Transformation of Terminators/Promoters

Transforming the following parts from the iGEM distribution for use in shuttle vector

BBa_B1006 from 2013 Plate 3, Well 6D (in C3)

BBa_Bl003 from 2013 Plate 3, Well 4P (in C3)

Punctured wells with pipette tip

Re suspended DNA in 10μL of PCR water

1μL of DNA added to 50μL of competent cells

100μL of cells  Used 2μL of DNA

Incubated on ice for 30 minutes

Heat shocked cells for 1 minutes

Incubated on ice for 5 minutes

Add 600μL of Psi broth

Incubated at 37°C with shaking for 2 hours

Also Xformed

BBa_K148012 Pveg
BBa_K823000 P?
BBa_K823002 P?
BBa_K823003 Pveg
BBa_K090504 ?

Plated 90μL of undiluted 1:10, 1:100, 1:1000 of BBa_B1006 and BBa_B1003

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07/22/13

PCR Cleanup of Cs Adh PCR Products

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07/22/13

Plasmid Extraction of Xformants

mlsR library colonies 5, 9, 10, 12, 22, 24

Extracted using Qiagen plasmid prep kit

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07/19/13

PCR of Ccel Adh

Preformed PCR to amplify Adh out of C. cellulolyticum

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07/19/13

PCR of Cb SAdh

Choose lower temperature (50°C) for annealing.

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Still have primer dimer, or primers may be sitting down at another place on the genomic DNA

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07/19/13

Promoter and Transformation Trouble Shooting

No colonies have grown on the plates incubated with transformed Top 10 cells with pSB1C3 and various promoters plated on July 16 2013. We did a nanodrop quantification of the ligationproducts.

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While the 260/280 ratios seem high, Dr. wawrick says that they aren’t troubling. However, the amount of DNA quantified is unrealistic. Since, we only started with 100ng of insert and 200ng of vector. We ran a gel to see if what we quantified is DNA and not RNA.

Well 1-Ladder

Well 2-Aadc – RBS

Well 3- thl + RBS

From this gel, it doesn’t look like we actually have any DNA (although there may be some really faint bands near the bottom). This means that the ligation didn’t actually occur (or we would have seen a band at 2000bp + 125-75bp). However, if the ligation didn’t work we would still expect to see 2 bands. One is the size of the vector and the other is the size of the insert. Perhaps, something went wrong in the phenol/chloroform extraction step.

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07/19/13

Restreak of mlsR + pSB1C3 Top 10

Transferred six colonies from library to individual plates. Numbers 5, 9, 10, 12, 22, and 24 from library.

PCR Purification of July 18, 2013 Cs

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07/18/13

PCR of C sacch and Cs Adh

Row 1
Lane Number	Sample	Dilution	Temperature (°C)
1	Ladder
2	Cs	Undiluted	55
3	Cs	Undiluted	62
4	Cs	Undiluted	65
5	Cs	1:10	55
6	Cs	1:10	62
7	Cs	1:10	65
8-16	Empty
Row 2
Lane Number	Sample	Dilution	Temperature (°C)
1	Ladder
2	Cb	Undiluted	55
3	Cb	Undiluted	62
4	Cb	Undiluted	65
5	Cb	1:10	55
6	Cb	1:10	62
Cb	Cs	1:10	65
8-16	Empty

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07/18/13

Gel of Isolated Plasmids

Row 1
Lane Number	Sample
1	Ladder
2	pSB1C3 + mlsR1
3	pSB1C3 + mlsR7
4	pSB1C3 + mlsR13
5	pSB1C3 + mlsR13
6	pSB1C3 + mlsR21
7	pSB1C3 + mlsR23
8	pSB1C3 + mlsR26
9	puc19 + KIVD

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07/18/13

Dual Digests of Isolated Plasmids (18 July)

500ng of plasmid for digest

puc19 + KIVD

500ng X (μL/516ng) = 0.89μL

pSB1C3 + mlsR21

500ng X (μL/99.4ng) = 5.03μL

pSB1C3 + mlsR13

500ng X (μL/81.0ng) = 6.17μL

pSB1C3 + mlsR1

500ng X (μL/101.0ng) = 4.95μL

pSB1C3 + mlsR7

500ng X (μL/65.1ng) = 7.68μL

pSB1C3 + mlsR23

500ng X (μL/84.1ng) = 5.95μL

pSB1C3 + mlsR26

500ng X (μL/86.1ng) = 5.81μL

Gene	EcoR1	Pst1	10x Buffer	DNA	PCR Water
KIVD	2μL	2μL	2μL	1μL	13μL
mlsR21	2μL	2μL	2μL	5μL	9μL
mlsR13	2μL	2μL	2μL	6μL	8μL
mlsR1	2μL	2μL	2μL	5μL	9μL
mlsR7	2μL	2μL	2μL	7.5μL	6.5μL
mlsR23	2μL	2μL	2μL	6μL	8μL
mlsR26	2μL	2μL	2μL	6μL	8μL

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07/18/13

Plasmid Prep of Transformed Top10 Cells with pSB1C3 + mlsR and puc19 + KIVD

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07/17/13

DNA Extraction from Ca, Cs, Cb, and Cc using MoBio Power Soil DNA Extraction Kit

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07/10/13

C. acetobutylicum Shuttle Vector Design

The following sequences have been verified:

Clos Ori BBa_J238001

Pthl BBa_J238002

Paadc BBa_J238003

Ptb BBa_J238004

mlsR BBa_J238005

Use Terminators for test testing

Long artificial terminator (T>90%)

BBa_B1006
39 base pairs
2013 p3w6D in C3

Small artificial terminator (T = 85%)

BBa_B1002
34 base pairs
2013 p5w4D in Ak3

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07/10/13

1% Agarose Gel Electrophoresis of 9 July PCR Products

Procedure followed as on pg. 50

Row 1
Lane	Sample	Dilution	Temperature (°C)
1	Ladder
2		C. sacc	1:10	50.8
3	C. sacc	1:100	50.8
4	C. sacc	1:1000	50.8
5	mlsR	1:10	50.8
6	mlsR	1:100	50.8
7	mlsR	1:1000	50.8
8	C beij	1:10	50.8
9	C beij	1:100	50.8
10	empty
11	empty
12	empty
13	empty
14	empty
15	empty
16	empty
Row 2
1	Ladder
2	C. sacc	1:10	52.8
3	C. sacc	1:100	52.8
4	C. sacc	1:1000	52.8
5	mlsR	1:10	52.8
6	mlsR	1:100	52.8
7	mlsR	1:1000	52.8
8	C beij	1:10	52.8
9	C beij	1:100	52.8
10	empty
11	empty
12	empty
13	empty
14	empty
15	empty
16	empty
Row 3
1	Ladder
2		C. beij	1:10	54.1
3	C. beij	1:100	54.1
4	mlsR	1:10	54.1
5	mlsR	1:100	54.1
6	mlsR	1:1000	54.1
7	C. sacc	1:10	54.1
8	C. sacc	1:100	54.1
9	C. sacc	1:1000	54.1
10	empty
11	empty
12	empty
13	empty
14	empty
15	empty
16	empty
Row 2
1	Ladder
2	C. beij	1:10	58.8
3	C. beij	1:100	58.8
4	mlsR	1:10	58.8
5	mlsR	1:100	58.8
6	mlsR	1:1000	58.8
7	C. sacc	1:10	58.8
8	C. sacc	1:100	58.8
9	C. sacc	1:1000	58.8
10	empty
11	empty
12	empty
13	empty
14	empty
15	empty
16	empty

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07/9/13

DNA Extraction for C sacc, C aceto, and C beij

Procedure followed as on pg. 50

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07/9/13

Gradient PCR of C. scc, mlsR, and C. beij

Column Number	Temperature (°C)
3	50
5	52.8
6	54.1
10	58.8

Lane Number	Sample
1	C sacc 1:10
2	C sacc 1:100
3	C sacc 1:1000
4	mlsR 1:10
5	mlsR 1:100
6	mlsR 1:1000
7	C beij 1:10
8	C beij 1:100

Vf Preparation

Component	Amount
2x Mastermix	125μL
Forward	2μL
Reverse	2μL
PCR dH₂O	121μL
Total	250μL

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07/3/13

Gel of (2 July) PCR Products

Row 1
Lane	Sample
1	Ladder
2		C. sacc 1:10
3	C. sacc 1:100
4	C. sacc 1:1000
5	C. sacc 1:10000
6	Negative
7	mlsR 1:10
8	mlsR 1:100
9	mlsR 1:1000
10	mlsR 1:10000
11	Negative
12	ladder
13	empty
14	empty
15	empty
16	empty
Row 2
1	Ladder
2	C. sacc 1:10
3	C. sacc 1:100
4	C. sacc 1:1000
5	C. sacc 1:10000
6	Negative
7	C. beij 1:10
8	C. beij 1:100
9	C. beij 1:1000
10	C. beij 1:10000
11	empty
12	empty
13	empty
14	empty
15	empty
16	empty

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07/2/13

PCR of mlsR and Redo of C. sacc and C. beij PCR

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07/2/13

Ralstonia eutropha DNA Extraction

Due to previous low extraction efficiency

Attempted to extract higher [DNA]

For 5mL tubes, each containing 3mL of meat extract broth inoculated with R. eutropha cells, which were in stationary phase
1mL aliquots transferred stepwise to two 15mL epi tubes, spinning at 14,000 rpm for five minutes to pellet cells
Supernatant discarded and additional 1mL aliquot added, spun until all cells pelleted
Cells re suspended in 500μL if 1x STE buffer in each of the two epi tubes
Vortex
50μL of 10% SDS added to each tube
500μL of TE saturated phenol pH8 added to each tube
Vortex well and centrifuge for 30 seconds
Aqueous recovered
Phenol extraction
Steps 7-9 repeated
500μL chloroform added to each tube and vortexed
Aqueous discarded
Chloroform extraction repeated
400μL split into each of four tubes
100μL of 3M NaOH added to each tube
350μL isopropanol added to each tube
Centrifuge at 14000 rpm for 25 minutes
Liquid discarded
Tubes places on 80°C heating block for 15 minutes
Contents of each tube re suspended in 500μL of PCR dH₂O

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07/2/13

Gel Electrophoresis of PCR Product (1 July)

Lane	Sample
1	Ladder
2		C. sacc Undiluted
3	C. sacc 1:10
4	C. sacc 1:100
5	Negative
6	C. cell undiluted
7	C. cell 1:10
8	C. cell 1:100

Lane	Sample
1	Ladder
2		Negative Control
3	1:10 Diluted C. beij
4	C. beij 1:100
5	C. beij 1:1000
6	C. beij 1:10000
7	Empty
8	Empty

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07/1/13

16S Gel Repeat

16S gel of C. acetobutylicum, R. eutropha, C. sacchaolyticum, and C. beijerinki to confirm wheather or not we have what we think we have

16S Gel
Lane Number	Sample
1	Ladder
2	C. beijerinki
3	R. eutropha
4	C. sacchrolyticum
C. acetobutylicum

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06/28/13

1:4 Loading Dye

1 part loading dye

2 parts 40% glycerol

2 parts PCR DI water

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06/27/13

DNA Extraction of R. eutropha, C. saccharolyticum, and C. beijerinki

Procedure performed as outline on pg. 42

16S PCR of C. acetobutylicum, R. eutropha, C. sacchrolyticum, and C. beijerinki to confirm wheather or not we have what we think

PCR Solution
Component	Amount (μL)
2x PCR Mastermix	350
Forward primer (27F)	Reverse primer (1525R
PCR water	344
Total	700

Undiluted
1:10
1:100

Place 1μL template into each tube

Negative control won’t have a template

Row Number	Colum Number	Sample
3	7	Negative Control
4	4	C. beijerinki
4	5	C. beijerinki 1:10
4	6	C. beijerinki 1:100
4	7	C. sacchrolyticum
4	8	C. sacchrolyticum 1:10
4	9	C. sacchrolyticum 1:100
5	4	C. acetobutylicum
5	5	C. acetobutylicum 1:10
5	6	C. acetobutylicum 1:100
5	7	R. eutropha
5	8	R. eutropha 1:10
5	9	R. eutropha 1:100

Gel of PCR product
Row Number	Colum Number	Sample
1	1	Ladder
1	2	C. beijerinki
1	3	C. beijerinki 1:10
1	4	C. beijerinki 1:100
1	5	C. sacchrolyticum
1	6	C. sacchrolyticum 1:10
1	7	C. sacchrolyticum 1:100
2	1	Ladder
2	2	C. acetobutylicum
2	3	C. acetobutylicum 1:10
2	4	C. acetobutylicum 1:100
2	5	R. eutropha
2	6	R. eutropha 1:10
2	7	R. eutropha 1:100
2	8	Negative Control

We have bands of the correct length for Cb, Cs, and Ca, they are too faint for sequencing. So, we are going to redo the PCR without template dilutions and do 35 cycles instead of 30 cycles

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06/25/13

Modified VM Medium

C. cellulolyticum may be grown at 37°L

VM medium to reduce precicpitation

AEM: 77, 2727-2733 (2011)

Modified ? (per liter)
Component	Amount
KH₂PO₄	1g
KH₂PO₄	3.4g
Urea	2.14g
MgCl₂ - 6H₂O	1g
CaCl₂ - 2H₂O
FeSO₂ - 6H₂	1.25g
MOPS 3-(N-morpholino)propanesulfuric acid	10g
Rezazurin	2mg
Vitamin solution	10mL
Yeast Extract	2g
Oligoelement solution	1mL
Cysteine – HCl	1g
Cellobiose	5.1345

100x Vitamin Solution
ComponentAmount
Biotin	0.08μM
Pyridoxamine	0.02μM
Cyanocobalamine	0.001μM
p-aminobenzoic acid	0.15μM
Thiamine	0.9μM
L-alaning	0.22μM

1000x Oligoelement Solution (per liter)
Component	Amount
FeSO₄ - 7H₂O	5g
ZnSO₄ - 7H₂O	1.44g
MnSO₄ - 7H₂O	1.12g
CuSO₄ - 5H₂O	0.25g
Na₂B₄O₇	0.2g
(Mo)₇(NH₄)₆O₂₄ - 4H₂O	1g
NiCl₂	0.04g
CoCl₂	0.02g
HBO₃	0.03g
Na₂SeO₃	0.02g
10M HCl	50mL

VM Medium placement

100mL in bottles
20mL in vials

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06/25/13

Re constituting Strains in Anaerobic Chamber

Clean glass vial with acetone
In aerobic Chamber:

Chemwipes
Sterile Needles

Bring into chamber

Heavy duty aluminum foil to work over
Glass cutter
File

Cycle chamber airlock; twice
Clean gloves and chamber with 1N HCl (x2)
???????????????????
Use sterile syringe to add 0.5mL media to inner vial
Mix with syringe
Add 0.5 of inoculums to each of the two media vials
Transfer an inoculum from these vials to a second vial to get one heavy and one light inoculum

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06/24/13

Media Preparation for C. acetobutylicum

Media components were added to a 2000mL Erlenmeyer flask

0.1mL of 1g/L Rezarin was added to the medium and was degassed under N₂ for 45 minutes

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06/21/13

Media Preparation for C. acetobutylicum

C. acetobutylicum will grow on defined media:

AEM 50:1238 (1985) or the following

Dr. Tanner’s recipe of C .acetobutylicum (per liter)
Component	Amount
Yeast Extract	1g
Glucose/Dextrose	20g
RST minerals	10mL
RST vitamins	10mL
RST metals	5mL

RST Minerals (per liter)
Component	Amount
NaCl	80g
NH₄Cl	100g
KCl	10g
KH₂PO₄	10g
MgSO₄ - 7H₂O	20g
CaCl₂ - 2H₂O	4g

RST Minerals (per liter)
Component	Amount
Nitriloacetic Acid	2g
MnSO₄ - H₂O	1g
Fe(NH₄)₂(SO₄)₂ - 6H₂O	0.8g
CoCl₂ - 6H₂O	0.2g
2nSO₄ - 7H₂	0.2g
CuCl₂ - 2H₂O	0.02g
NiCl₂ - 6H₂O	0.02g
Na₂MoO₄ - 2H₂O	0.02
Na₂SeO₄	0.02g
Na₂WO₄	0.02g

RST Vitamins
Components	Amount	Light Sensitivity (Y/N)
Pyridoxine HCl	10mg	Y
Thiamine HCl	5mg	Y
Riboflavin	5mg	Y
Calcium Pantothenate	5mg	Y
Thioctic Acid	5mg	N
p-Aminobenzoic Acid	5mg	Y
Nicotinic Acid	5mg	N
B₁₂ = Cyanocobalamine	5mg	N
Biotin	2mg	N
Folic Acid	2mg	Y
Mercaptopethane Sulfuric Acid	10mg	N

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06/17/13

Competent pAN1 containing DH5 E. coli

Suspended a unit sized inoculums of pAN1 containing DH5 E. coli in LB with chloramphenicol resistance.

Incubated overnight at 37°C and at 220 rpm for 2.5 hours

One culture has an OD600 of 0.38 and another is 0.475

Spun at 12000 g for 20 minutes
Discarded supernatant and re suspended in 15mL of TFBl
Incubated on ice for 60 minutes
Centrifuged at 12000 g for 20 minutes

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06/14/13

Gel of puc19 with KIVD Digest (EcoR1 and Pst1)

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06/14/13

Digestion of pUC19 containing KIVD with EcoR1-Hf and Pst1-Hf

Component	Amount (μL)
EcoR1	1
Pst1-Hf	1
10x Buffer	2
DNA	2.5 (500ng)
PCR H₂	13.5
Total	20

Place in 37°C water bath for 45 minutes

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06/13/13

Gel of PCR

Row 1
Well Number	Sample	Temperature (°C)
1	Ladder
2	AdhE1	50.8
3	AdhE1	52.8
4	AdhE1	54.1
5	AdhE1	58.8

Row 2
Well Number	Sample	Temperature (°C)
1	Ladder
2	AdhE	50.8
3	AdhE	52.8
4	AdhE	54.1
5	AdhE	58.8

The gel results of this PCR are similar to the last one. The bands indicate smaller than 250 base pairs, which is smaller than the size of the genes we intended to amplify (2000 base pairs).

AdhE = 2589 base pairs

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06/12/13

PCR redo of AdhE and AdhE1

Temperature gradient and dilution up to 1:10000

Column Number	Temperature (°C)
3	50.8
5	52.8
6	54.1
10	58.8

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06/12/13

Plasmid Isolation of pUC19 from BBa_k273006

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06/10/13

Amplification of AdhE and AdhE1 in C. acetobutylicum

Testing our primers using the template prepared on June/4

Forward primer used (AdhE)

Forward primer sequence

Reverse primer used (AdhE)

Reverse primer Sequence

Forward primer used (AdhE1)

Forward primer sequence

Reverse primer used (AdhE1)

Reverse primer sequence

Preparation of solution 1:

Into two separate 1.5mL centrifuge tubes

Component	Amount (μL)
PCR DI Water	146
2x PCR Mastermix	150
Forward primer	2
Reverse primer	2
Total	300

50μL of solution 1 were pipette into 5 PCR tubes for each solution (AdhE and AdhE1)

Template dilutions were prepared for 1:10, 1:100, 1:1000, and 1:10000

1mL of template was loaded into 2 each of 4 PCR tubes containing 50μL of solution 1

The 5th PCR tube containing solution 1 will serve as negative control

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06/4/13

Place pxy plates in glove box to degrass

Extracted Ca DNA

Re suspend cells in 500μL 1x STE buffer
Vortex
Add 1/10 volume of 10% SDS
2 minutes in bead beater
Add 500μL TE saturated phenol pH 8

Be sure to go down far enough in bottle to reach phenol
Put directly back in fridge when done

Vortex
Centrifuge for 30 seconds
Recover aqueous phase

Don’t recover white material at interphase
Throw away

Add 500μL Chloroform: Isoamyl acetate
Vortex
Centrifuge for 30 seconds
Recover and throw away aqueous phase
Repeat Steps 8-12
Add 2/10 volumes 3M sodium acetate
Add 7/10 volumes isopropanol
Mix well
Centrifuge for 25 minutes
Remove liquid and don’t disturb invisible pellet
Dry pellet at 60°C in heat block for 10 minutes

Leave caps off to allow isopropanol to evaporate

Re suspend pellet in 50μL of dH₂

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05/31/13

Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin

Digestion as preformed on pg. 47

Gel of Digestion
Row 1
Well Number	Sample
1	Ladder
2	1
3	3
4	5
5	8
6	11
7	12
Row 2
Well Number	Sample
1	Ladder
2	16
3	18
4	20
5	22
6	24

Wells 2 and 3 like like what we need.

Sequencing primers are in the process of being made to comfirm

Row 2
Well Number	Sample	Temperature (°C)
1	Ladder
2	AdhE	50.8
3	AdhE	52.8
4	AdhE	54.1
5	AdhE	58.8

The gel results of this PCR are similar to the last one. The bands indicate smaller than 250 base pairs, which is smaller than the size of the genes we intended to amplify (2000 base pairs).

AdhE = 2589 base pairs

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06/12/13

PCR redo of AdhE and AdhE1

Temperature gradient and dilution up to 1:10000

Column Number	Temperature (°C)
3	50.8
5	52.8
6	54.1
10	58.8

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06/12/13

Plasmid Isolation of pUC19 from BBa_k273006

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06/10/13

Amplification of AdhE and AdhE1 in C. acetobutylicum

Testing our primers using the template prepared on June/4

Forward primer used (AdhE)

Forward primer sequence

Reverse primer used (AdhE)

Reverse primer Sequence

Forward primer used (AdhE1)

Forward primer sequence

Reverse primer used (AdhE1)

Reverse primer sequence

Preparation of solution 1:

Into two separate 1.5mL centrifuge tubes

Component	Amount (μL)
PCR DI Water	146
2x PCR Mastermix	150
Forward primer	2
Reverse primer	2
Total	300

50μL of solution 1 were pipette into 5 PCR tubes for each solution (AdhE and AdhE1)

Template dilutions were prepared for 1:10, 1:100, 1:1000, and 1:10000

1mL of template was loaded into 2 each of 4 PCR tubes containing 50μL of solution 1

The 5th PCR tube containing solution 1 will serve as negative control

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06/4/13

Place pxy plates in glove box to degrass

Extracted Ca DNA

Re suspend cells in 500μL 1x STE buffer
Vortex
Add 1/10 volume of 10% SDS
2 minutes in bead beater
Add 500μL TE saturated phenol pH 8

Be sure to go down far enough in bottle to reach phenol
Put directly back in fridge when done

Vortex
Centrifuge for 30 seconds
Recover aqueous phase

Don’t recover white material at interphase
Throw away

Add 500μL Chloroform: Isoamyl acetate
Vortex
Centrifuge for 30 seconds
Recover and throw away aqueous phase
Repeat Steps 8-12
Add 2/10 volumes 3M sodium acetate
Add 7/10 volumes isopropanol
Mix well
Centrifuge for 25 minutes
Remove liquid and don’t disturb invisible pellet
Dry pellet at 60°C in heat block for 10 minutes

Leave caps off to allow isopropanol to evaporate

Re suspend pellet in 50μL of dH₂

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05/31/13

Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin

Digestion as preformed on pg. 47

Gel of Digestion
Row 1
Well Number	Sample
1	Ladder
2	1
3	3
4	5
5	8
6	11
7	12
Row 2
Well Number	Sample
1	Ladder
2	16
3	18
4	20
5	22
6	24

Wells 2 and 3 like like what we need.

Sequencing primers are in the process of being made to comfirm

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05/30/13

Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin

Purified six tubes of DNA (plasmid)

Quantification with Nanodrop

Digestion
Component	Amount (μL)
EcoR1	2
Pst1	2
10x Buffer	1
5

Incubate in 37°C water bath for 1 hour

Load all 10μL onto gel

Gel of Digestion
Well Number	Sample
1	Ladder
2	Screen #3
3	Screen #6
4	Screen #10
5	Screen #19
6	Screen #19
7	Screen #26

Note: Screen # corresponds to quantification Id

Since our insert is about 700 base pairs and our vector is about 2000 base pairs. Well 2 looks like what we want. We are going to re streak colonies isolated from plate with screen #3 , make a freezer stock, and take more colonies from screen #10

Freezer stocks of Top 10, pAN1, pIKM1, and p34KM were made by suspending cells in 1mL of 10% glycerol.

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05/29/13

Made 80 stocks of C. acetobutylicum (2mL of culture into 2mL of glycerol)

Streaked three more Top 10 pSB1C3 and Clostridial Origin plates

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05/28/13

Inoculated cultures of C. acetobutylicum and a transfer was made

Six plates (LB + Chloramphenicol) were streaked with transformation of pSB1C3 and Clostridial Origin in Top 10 E. coli cells in preparation for plasmid extraction.

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05/23/13

After 824: possible growth
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05/22/13

Re Dual Digest of pSB1C3 and Insert

Procedure as preformed on pg. 38

Gel of pSB1C3 and Insert Digestion
Well Number	Sample
1	Ladder
2	pSB1C3
3	Clostridial Origin

Gel of pSB1C3 and Insert Digestion after Quanitification
Well Number	Sample
1	Ladder
2	pSB1C3
3	Clostridial Origin

Ligation:

Insert, Vector: 21μL
Ligase Buffer: 5μL
Ligase: 3&#956:L

Let reaction sit on bench for 90 minutes

OR

For overnight ligation: 14°C water bath

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05/9/13

Repeat of Dual Digest of pSB1C3 and Clostridial Origin Fragment

We’re repeating this procedure, due to that fact that days between a digestion and ligation shows to be ineffective. Hence, a ligation should be completed soon after digestion.

pSB1C3
Component	Amount (μL)
EcoR1	2
Pst1	2
10x Buffer	2
DNA	2.5
PCR H₂	11.5
Total	20

Clostridial Origin Fragment
Component	Amount (μL)
EcoR1	2
Pst1	2
10x Buffer	2
DNA	10
PCR H₂	4
Total	20

Place in 37°C water bath for 15 minutes

Ran gel

Well Number Sample 1 Ladder 2 Clostridial Origin 3 pSB1C3

Used wrong kit (Spin mini prep). Suppose to use PCR purification kit.

\

Cleaned/purified digests

Clostridial Origin

pSB1C3

Ligation:

5μL 10x Buffer
2μL ligase (quick)
?μL vector
?μL insert
Up to 50μL dH₂

[(ng vector X size insert in Kb)/Size vector in Kb] X molar ratio of insert : vector = ng of insert

Since we used the wrong kit, we’re going to precipitate the DNA out (used digestion on May/9)

Phenol Chloroform Protocol pH 8:

Turn on heating block
Add equal amounts of phenol and whatever volume DNA is in (20μL)

Phenol : Pipette below top surface

Vortex
Centrifuge for 30 seconds

Two layers appear
Protein is inactive and at interphase
Pipette out top layer of both and combine aqueous

Need to get rid of phenol because it absorbs same wavelength as does water and interferes with enzyme activity

Add 50μL of CIAA 24:1

Removes Phenol

Vortex
Centrifuge for 30 seconds
Remove top layer (aqueous)

Leave a little behind so not to contaminate aqueous phase with bottom layer

Add 50μL of CIAA 24:1
Vortex
Centrifuge for 30 seconds
Remove top layer (aqueous)

Leave a little behind so not to contaminate aqueous phase with bottom layer

Now we have 50μL of DNA

Add 1/10 volume sodium acetate 3M pH 5.2
Add 7/10 volume (total) of isopropanol
Vortex
Centrifuge for 20 minutes at 14,000 rpm
Dispose of liquid, leaving pellet inside tube
Add 50μL of 10% ethanol to get rid of isopropanol
Centrifuge for 30 seconds
Dispose of liquid
Use heat block to dry

60°C for 15 minutes
Leave tube open

Add 10μL of PCR water to re suspend pellet
Use 2μL to quantify via nandrop
Add 1μL Ligase (quick)
Add 1μL 10x Buffer = 10μL reaction
Let reaction sit at room temperature for 2 hours

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05/9/13

Dual Digest of pSB1C3 and Clostridial Origin Fragment

pSB1C3 = 2000 base pairs

Costridial Origin = 700 base pairs

Want to digest 500ng of backbone and 1:1 ratio of backbone to insert

pSB1C3 linearized backbone = 202.1 ng/μL

500ng of pSB1C3: $500ng x μL/202ng = 2.5μL$

1:1 mole ratio of backbone to insert

500ng x (2000 bp/mol /700 bp/mol)  = 175ng insert

175ng of insert:

175ng x μL/17ng  = 10μL

pSB1C3
Component	Amount (μL)
EcoR1	2
Pst1	2
10x Buffer	2
DNA	2.5
PCR H₂	11.5
Total	20

Clostridial Origin Fragment
Component	Amount (μL)
EcoR1	2
Pst1	2
10x Buffer	2
DNA	10
PCR H₂	4
Total	20

Place in 37°C water bath for 15 minutes

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05/9/13

PCR Cleanup of Linearized Backbone of Quantification

Used QIAquick PCR Cleanup Kit and processed pSB1K3 and pSB1A3 linearized plasmid backbone PCR products

Nanodrop Quantification

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05/9/13

Gel of New Linearized Backbone using Provided Supermix

Well Number	Sample
1	Ladder
2	pSB1K3 (blue Hp) (fail: pipette tip fell into well)
3	pSB1K3
4	pSB1A3 (blue Hp)
5	pSB1A3

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05/8/13

Linearized Backbone PCR

Dilute plasmid backbones to 10ng/μL

Two reactions for pSB1K3 and pSB1A3
Component	Amount (μL)
PCR High Fidelity Supermix	97
Primer 1: SB prep 3P-1	1
Primer 2: SB prep 2Ea	1
Template	10ng

pSB1K3

10ng x μL/75ng  = 1 : 7.5μL Dilution     10μL : 65μL PCR H

₂O

pSB1A3

10ng x μL/40ng = 1 : 4μL Dilution     10μL : 30μL PCR H

₂O

Thermo Cycler (x38)

95°C for 2 minutes
95°C for 30 seconds
55°C for 30 seconds
68°C for 3 minutes
69°C for 10 minutes

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02/12/13

stuff stuff stuff stuff

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05/1/13

PCR Supermix High Fidelity Preparation

To have eight reaction vials with 50μL supermix each. Extra is made for potential pipetting error.

Component	Amount (μL)
Amplification Buffer 10x	50
dNTP Mixture (10 mM)	15
50 mM MgSO₄	10
Pfx DNA Polymerase (Hf) (Spin down)	10
dH₂O	415
Total (two different backbones)	500

PCR reaction of linearized backbone in each tube
Component	Amount (μL)
PCR Supermix HF	50
SB prep 3P-1	0.4
SB prep 2Ea	0.4
DNA	5ng

pSB1K3

5ng x μL/28ng  =0 .18μL  => 0.2μL

pSB1A3

5ng x μL/135.8ng& = 0.037μL => 0.04μL

For our eight reactions, multiply all values (except supermix) above by 10 and add/mix into 250μL of supermix

Final Reaction Tube Composition
Component	Amount (μL)
PCR Supermix	250
SB prep 3P-1	4
SB prep 2Ea	4
pSB1K3 DNA	50ng = 2μL
pSB1A3 DNA	50ng = 0.4μL

Alliquot 55μL of supermix , primers, and template into each tube

To prepare dNTP mix

10μL of each NYP
60μL of dH₂O

PCR for linearized backbone (actual)

Four reaction of 1 backbone
Component	Amount (μL)
10x Buffer	25
Polymerase Hf (spin)	5
Mg	5
dNTP mix	7.5
DI H₂O	203
Forward Primer	2
Reverse Primer	2
Template	1 (may need to dilute)
Total	250

To dilute pSB1A3:(135.8 ng/μL)

1μL Template

4μL PCR dH₂O

We didn’t get what we were trying to amplify

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04/28/13

PCR Reaction with Primer Set #3

Preformed as on pg. 15 with the following modifactions

Dilutions
- 1:10
- 1:100
- 1:1000

95°C for 45 seconds
52°C for 45 seconds
72°C for 2 minutes

Repeat 30x

32ng/μL of DNA template (pIKM1) successfully produced the Clostridial origin

PCR product was digested using QIAquick PCR purification kit

Note: If solution turns pick, the pH needs to be adjusted with 5μL of sodium acetate

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04/24/13

Anaerobic Media Preparation

For 0.5L

500mL dH₂
15g TSB mix
1g glucose
Adjust pH to 7.3
Place sponge stopper in place
Open silver valve and black valve
Set degassing station to 20 psi
Switch to nitrogen and run for 30 seconds to flush oxygen out of head space
Place largest needle into media and turn on spin located on stir-plate, then set time for 45 minutes

Disinfect top of bottle with alchol wipe
Add sterile wipe filter with new needle
Need steady stream of bubbles

Add 0.1mL Resasrin (will be purple until autoclaved)
Post autoclaved

Pink color signifies media has been exposed to oxygen
No color in media signifies media is anaerobic

Oxygen scavenger
If black precipitant forms after autoclave and before transfer into media bottles, then too much oxygen present.

Use acid washed vials
Place stoppers in ice and water to shrink pores
Flush bottles and vials with nitrogen
Place 35mL in each using automated pipette
Angle stopper with needle (gas still within)
Leave for 10 seconds
Bring needle out of bottle and push stopper in
Crimp top
Add 15mL to vials
Flush head space

Put regular needle in and flushing needle in
Flush for 3 minutes
Pull both needles out at same times so you don’t put pressure on tubes

OR

Switch degassing station to vacuum
Alternate between vacuum and pressure 10 times

Let it settle back to position before going back to pressure

Make sure all needles are in black stopper
Tubes now have 20 psi and poke with needle to vent
Turn off tank
Autoclave immediately

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04/19/13

PCR of Clostridial Origin with New Primers

95°C for 45 seconds
52°C for 45 seconds
72°C for 2 minutes

Repeat 30x

well 1: 1:10 dilution of template
well 2: 1:100 dilution
well 3: 1:1000 dilution
well 4: negative control

The band we get from the gel is too small to be the origin that we want. We think that we have just been given something other than what we thought we had

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04/19/13

Making Bulk Amounts of Linearized Plasmid Backbones

PCR Supermix High Fidelity	9.6mL
Primer SB-prep 2Eb	65μL
Primer SB-prep 3P-1	65μL
DNA Template	100ng

pSB1K3

100ng x μL/40ng  = 9.8μL  => 2.5μL + 7.5μL dH

₂

pSB1A3

100ng x μL/75ng& = 1.6μL => 1.5μL + 8.5μL dH

₂

pSB1C3

100ng x μL/43ng& = 1.6μL => 2.5μL + 7.5μL dH

₂

High fidelity aliquots of 100μL for PCR

PCR

95°C for 2 minutes
95°C for 30 seconds
55°C for 30 seconds
68°C for 3 minutes
68°C for 10 minutes

PCR Cleanup: Use Quiagen

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04/9/13

Gel of 8 Apr 2013 Digest Purifications

Row 1
Lane	Sample
1	1 Kb Plus Ladder
2	pIKM1 + RSA1 digest
3	pIKM1
4	pAN1 + RSA1 digest
5	pAN1

85 Volts for 1 hour

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04/8/13

Verification of pIKM1/pANi

Because we may have accidently stimulated the plasmid and mislabeled them previously. Since, no digestion of the plasmid pIKM1 from last weeks experiment is consistent with a methylating plasmid, today we are digesting both plasmids and running a gel.

This should verify that either we mixed up our plasmids, or we received the wrong plasmid.

1) Plasmid preparation of pAN1 and pIKM1 via Qiagen QiaPrep Spin Miniprep Unit instructions

2) Nanodrop quantification of plasmid DNA

3) Digest of pIKM1 and pAN1 with RSA1

pAN1

500ng;times;μL/51ng  = 9.8μL  => 10.0μL

pIKM1

500ng;times;μL/320ng& = 1.6μL => 2μ

Compnents	pAN1	pIKM1
DNA	10μL	2μL
RSA1	2μL	2μL
10x Buffer	2μL	2μL
PCR Water	6μL	14μL
TOTAL	20μL	20μL

Placed in 42°C water bath for 15 minutes

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04/5/13

Row 1
Lane	Sample
1	1 Kb Plus Ladder
2	pSB1C3 + RSA1 + Xba + Invitrogen Buffer
3	pSB1C3 + RSA1 + Xba + Fermentas Buffer
4	pSB1C3 + RSA1 + Xba + Invitrogen Buffer + Fermentas Buffer
5	pSB1A3 + RSA1 + Xba + Invitrogen Buffer
6	pSB1A3 + RSA1 + Xba + Fermentas Buffer
7	pSB1A3 + RSA1 + Xba + Invitrogen Buffer + Fermentas Buffer
8	PIKM1 + RSA1
9	pSB1K3 + Hind1 + Xba
10	Negative Control
11	PIKM1
12	pSB1K3

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04/3/13

Since we couldn’t find information about the Fermentas buffer needed for Xba1 and Hind3, we are going to set up our experiment in such a way that tests to see if using only the buffer from Fermentas, using the buffer from Invitrogen, or using a 1:1 mixture of the two will allow our digestion to occur in reactions where enzymes from different companies are being used.

Tubes will be labeled with the plasmid name, which enzyme used, and which buffer was used. For example

pSB1C3
RSA1 and Xba1
2μL of Invitrogen buffer

pSB1C3
RSA1 and Xba1
2μL of Fermentas buffer

pSB1C3
RSA1 and Xba1
1μL of Fermentas buffer and 1μL of Invitrogen buffer

We incubated digests in 42°C water bath for 15 minutes

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04/1/13

Restriction Digest of Biobrick and PIKM1 with AluI

PPIKM1

5μL Plasmid
2μL RSA1
2μL 10x Buffer
11μL PCR Water

Biobrick

5μL Plasmid
2μL RE1(XbaI)
2μL RE2(Hind 3 or RSA1
2μL 10x Buffer
9μL PCR Water

Use a gel of 2% agarose

0.8g of agarose
40mL of TAE 1x

Regarding a digest, you typically want to match the company that produced the enzyme you’re using to the same company for the buffer. If they’re not the same, look up components and concentrations.

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03/28/13

This looks to be the same size no matter the temperature. We hypothesize that either our primers are laying down non-specifically or they are oriented in the wrong direction. If it turns out our primers are fine, then we may have a different organis, than expected.

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03/27/13

PCR repeat of Clostridial origin repeat

Preformed as outline on page 15 with the following modifications

Only a 1:100 dilution was used since our DNA template had a concentration of 164.6 ng/μL, which is a good value to use for genomic DNA.
A temperature gradient for the annealing step was setup where different columns in thermocycler are a different temperature during the annealing process.

Column	Temperature (μC)
2	50.2μC
3	50.8μC
4	51.7μC
5	52.8μC
6	54.1μC
7	55.4μC
8	56.7μC
9	57.9μC
10	58.8μC
11	59.5μC

To set up temperature gradient on thermocycler

Highlight stage of interest (in this case; annealing)
Selection "options"
Select "Show Gradient"

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03/25/13

Gel of Clostridial Origin PCR Product

PCR analysis was completed March 13, 2013

well 1:Ladder
well 2:10 Dilution
well 3:100 Dilution
well 4:1000 Dilution
well 5:10000 Dilution
well 6:Control

From these gels, we can see a band between 250 and 500 bases, which isn't the size of our clostridial origin. Assuming PCR worked correctly, we should see a band of approximately 1200 base pairs. Since we ran multiple cells with the same result, we are hypothesizing that an error was in the PCR reaction. Therefore, we are going to repeat PCR before we move forward by adjusting the annealing temperature.

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03/15/13

Quantification of PCR Product and Linearized Vector with Chloramphenicol

Nanodrop Protocol

Click icon with ND-1000 on computer
Click "Nucleic Acids"
Wipe off pedestal with chemwipe
Load 3°L DI water to initialize and click blank
Click "Measure" to verify flat line
Load 3°L of sample
Click "Measure"
Click "Print" screen after sample ID has been typed in

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03/13/13

Making and Preparing Agarose Gel

In a 125mL erlenmyer flask, combine 0.4g ultra pure agarose and 40mL 1x TAE buffer, which makes a 1% gel
Swirl to mix. Place in microwave for 40 seconds. If all agarose isn't dissolved, heat again in 7 second increments
Run flask bottom under water to cool agarose
Pour into gel rig with comb inserted
Let gel cool until opaque

Running Gel of Post-Digested pSB1C3 and pSB1A3

Move gel into gel rig container, pour 1x TAE buffer until it covers the gel surface
Remove comb slowly
Mix 5-10μL of digest with 3μL of 1:4 EZ Vision dye (Note: if using undigested DNA, only use 2μL
Load samples into wells after 1 kb DNA ladder is loaded into far left lane

well 1:Ladder
well 2:pSB1C3 EcoR1
well 3:pSB1C3 EcoR1 Pst1
well 4:pSB1C3 Pst1
well 5:pSB1A3 EcoR1
well 6:pSB1A3 EcoR1 Pst1
well 7:pSB1A3 Pst1
well 8:pSB1C3 UNDIGESTED

Run gel for 45 minutes-1.5 hours at 85 volts

Run gel for 45 minutes-1.5 hours at 85 volts

PCR Reaction Protocol

Combine the following
- 150μL 2x master mix (polymerase,buffer)
- 1μL forward primer
- 1μL reverse primer
- 148μL DI water (PCR water, UV prior to use)
Make 1:10, 1:100, 1:1000, 1:10000 dilutions of template
In four tubes, combine 50μL of step 1 solution and 1μL of diluted template
in fifth tube, only put in step 1 solution as a negative control

Regular PCR Cycle

95°C for 45 seconds
55°C for 1 minute
75°C for 1.5 minutes
These three steps are cycled 35 times

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03/12/13

Preformed the following restriction digest of pSB1A3 and pSB1C3.

500ng DNA in 20μL

pSB1A3 [DNA] = 135.8ng/μL

pSB1C3 [DNA] = 74.7ng/μL

500ng x μL/135.8μg = 3.68μL

500ng x μL/74.7μg = 6.69μL

Sample	EcoRI	PstI	10X Buffer	DNA	PCR Water	TOTAL
pSB1A3	2μL	0μL	2μL	4μL	12μL	20μL
pSB1A3	2μL	2μL	2μL	4μL	10μL	20μL
pSB1C3	2μL	0μL	2μL	7μL	9μL	20μL
pSB1C3	2μL	2μL	2μL	7μL	7μL	20μL

Smiley face

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03/8/13

Nanodrop DNA Quanitification

Protocol

Open Nanodrop 7000 V.6.0
Select Nucleic Acid
Blank via 3μL of PCR water
Verify 0ng/&#;L DNA in PCR water
Load 3μL of sample
Record DNA concentration in ng/μL
Print Screen

pSB1K3

p34KM

pIKM1

pSB1A3

pSB1C3

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02/13/13

Colonies were counted

1:1000 dilution of Ampicillin^R pSB1A3 wasn't properly plated

Dilution	Plasmid	Colony Count	Cells/μg DNA
1:1000	pSB1C3	58	8.24e6
1:1000	pSB1K3	79	1.12e7
1:1000	p34KM	157	2.22e7
1:100	pSB1A3	1521	2.16e7

Note: 1:100 estimates by counting colonies in 1/3 of plate and multiplying by 3

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02/12/13

Plates were removed from 37°C incubator, wrapped in parafilm, and stored in 4°C refrigerator overnight

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02/13/13

pSB1C3

Dilution 1:1000

58 Colonies

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02/11/13

Transforming Competent Cells

We transformed TOP10 chemically competent cells using plasmids.

Resistance	Plasmid ID	Original Concentration (ng/μL)
Kanamycin	pSB1K3	62
Kanamycin	p34KM	40
Chloramphenicol	pSB1C3	43
Ampicillin	pSB1A3	75

Protocol

TOP10 competent cells in 100μL alliquots (x5) were thawed on ice and resuspended.
100ng of plasmid were added to cells
Cells were placed on ice for 20 minutes
Cells were transformed to a 42°C waterbath for 60 seconds
After 60 seconds in waterbath, add 600μL of Psi proth IMMEDIATELY to clls
Cells were incubated for 60 minutes at 37°C while shaking at 200rpm
Dilutions of transformation mixture were made at 1:10, 1:100, and 1:1000
50mL of each dilution was plated on an LB + Antibiotic plate
Plates were incubated at 37°C overnight

Plasmids were diluted to 20μL of 10μg/μL

p34KM

10ng/μL×μL/62ng×20μL = 3.2μL (original plasmid concentration) + 16.8μL (dH

₂O)

pSB1C3

10ng/μL×μL/43ng×20μL = 4.65μL (original plasmid concentration) + 15.35μL (dH

₂O)

pSB1A3

10ng/μL×μL/75ng×20μL = 2.6μL (original plasmid concentration) + 17.4μL (dH

₂O)

pSB1K3

10ng/μL×μL/40ng×20μL = 5.0μL (original plasmid concentration) + 15.0μL (dH

₂O)

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Buffers for Preparing Competent E. coli

02/4/13

TFB1: pH: 5.8/ Sterile Filter

Chemicals	Concentration (mM)
RbCl	100
MnCl₂	50
Potassium Acetate	30
CaCl₂	10
Glycerol	15% by Weight

TFB2 ph:6.8 (Use KOH to adjust)/ Sterile Filter

Chemicals	Concentrations (mM)
MOPS	10
RbCl	10
CaCl₂	75
Glycerol	15% by Weight

TFB1

Chemicals	Mass (g)
RbCl	3.02965
MnCl₂	1.56996
Potassium Acetate	0.74392
CaCl₂	0.27806
Glycerol	37.5463

TFB2
Chemicals	Mass (g)
MOPS	0.53398
RbCl	0.30225
CaCl₂	2.88320
Glycerol	37.5457

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Making Antibiotic Stocks

02/1/13

Antibiotics are used to isolate organisms into which plasmids containing antibiotic resistance genes have been transformed.

Make antibiotic plates with the following specs.

Antibiotic	Concentration (µg/mL)	Color Code
Ampicillin	100	Orange
Chloramphenicol	35	Green
Kanamycin	50	Red
Tetracycline	15	Yellow

Stocks should be made at 1000x concentration, so that making 1L of plate medium will require only 1mL of stock antibiotic solution.

Stocks of antibiotics are made at the following concentrations

Ampicillin	100 mg/mL
Chloramphenicol	35 mg/mL
Kanamycin	30 mg/mL
Tetracycline	15 mg/mL

50mL of stock were prepared and split into several 15mL tubes

Stocks should be stored in the refrigerator at 4°C

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01/25/13

Restreaked TOP10 cells on LB plates for isolation of single colonies.

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01/23/13

Sealed TOP10 E. coli cells on LB agar plate. Stored in 37°C incubator

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01/18/13

Poured LB agar plates

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01/16/13

Making Agar Plates

LB agar is used to grow our stocks of Escherichia coli

Recipe: Per 1000 mL

10g Bacto Tryptone
5g Yeast Extract
10g Sodium Chloride (NaCl)
15g Agarose

Mix Components in 1L of dH₂O until dissolved. Spilt 1000mL solution into two flask. Cap flask with aluminum to prevent spilling of solution. Autoclave on slow exhaust for 20 minutes. Keep liquid in 65°C water bath to prevent setting until 18-Jan.

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@@ Line 7: / Line 7: @@
 <div>
-<!--
+<h1></h1>
+<p class = "date">09/13/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of Pptb-</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row 1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder (1kb plus)</th>
+<tr>
+<th>2</th>
+<th>Pptb- #9A</th>
+<tr>
+<th>3</th>
+<th>Pptb- #9 B</th>
+<tr>
+<th>4</th>
+<th>Empty</th>
+<tr>
+<th>5</th>
+<th>Empty</th>
+<tr>
+<th>6</th>
+<th>Empty</th>
+<tr>
+<th>7</th>
+<th>Empty</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">09/13/13</p>
+<div align="center" style="border:1px solid white"><b>Digest of Pptb- Sample</b></div></br></br>
+<math>500ng X (&#956;L/178.1ng) = 2.81&#956;L</math>
+</br>
+<math>500ng X (&#956;L/110.1ng) = 4.54&#956;L</math>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Sample</th>
+<th>EcoR1</th>
+<th>Pst1</th>
+<th>10x Buffer</th>
+<th>DNA</th>
+<th>PCR dH<sub>2</sub>O</th>
+<th>Total</th>
+<tr>
+<td>Pptb- #9A</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2.81&#956;L</td>
+<td>11.19&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #9B</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.54&#956;L</td>
+<td>9.46&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+</table>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">09/11/13</p>
+<div align="center" style="border:1px solid white"><b>Quantification and DNA Extraction of 3A</b></div></br></br>
+<p>QIAprep Spin Miniprep Kit (250)
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/d/d6/92.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">09/10/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of Pptb- + Clos Ori</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row #1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<td>1</td>
+<td>Ladder (1kb plus)<td>
+<tr>
+<td>2</td>
+<td>Pptb- #1</td>
+<tr>
+<td>3</td>
+<td>pptb- #2</td>
+<tr>
+<td>4</td>
+<td>Pptb- #3</td>
+<tr>
+<td>5</td>
+<td>Pptb- #4</td>
+<tr>
+<td>6</td>
+<td>Pptb- #6</td>
+<tr>
+<td>7</td>
+<td>Empty</td>
+<tr>
+<td>8</td>
+<td>Empty</td>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/e/ec/91.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<p>Expected insert size of 125bp</p>
+<p>No valid inserts found</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">09/11/13</p>
+<div align="center" style="border:1px solid white"><b>Digest of Pptb-</b></div></br></br>
+<p>Pptb- #1</p>
+<math>500ng X (&#956;L/81.1ng) = 6.17&#956;L</math>
+</br>
+<p>Pptb- #2</p>
+<math>500ng X (&#956;L/86.4ng) = 5.79&#956;L</math>
+</br>
+<p>Pptb- #3</p>
+<math>500ng X (&#956;L/70.9ng) = 7.05&#956;L</math>
+</br>
+<p>Pptb- #4</p>
+<math>500ng X (&#956;L/147.7ng) = 3.39&#956;L</math>
+</br>
+<p>Pptb- #6</p>
+<math>500ng X (&#956;L/169.3ng) = 2.95&#956;L</math>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Sample</th>
+<th>EcoR1</th>
+<th>Pst1</th>
+<th>10x Buffer</th>
+<th>DNA</th>
+<th>PCR dH<sub>2</sub>O</th>
+<th>Total</th>
+<tr>
+<td>Pptb- #1</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>6.17&#956;L</td>
+<td>9.83&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #2</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>5.79&#956;L</td>
+<td>8.21&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #3</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>7.05&#956;L</td>
+<td>6.95&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #4</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>3.39&#956;L</td>
+<td>10.61&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #5</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2.95&#956;L</td>
+<td>11.05&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+</table>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">09/11/13</p>
+<div align="center" style="border:1px solid white"><b>Quantification and DNA Extraction of 3A</b></div></br></br>
+<p>QIAprep Spin Miniprep Kit (250)
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">09/10/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of Pptb- + Clos Ori</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row #1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<td>1</td>
+<td>Ladder (1kb plus)<td>
+<tr>
+<td>2</td>
+<td>Pptb- #1</td>
+<tr>
+<td>3</td>
+<td>pptb- #2</td>
+<tr>
+<td>4</td>
+<td>Pptb- #3</td>
+<tr>
+<td>5</td>
+<td>Pptb- #4</td>
+<tr>
+<td>6</td>
+<td>Pptb- #5</td>
+<tr>
+<td>7</td>
+<td>Pptb- #7</td>
+<tr>
+<td>8</td>
+<td>Pptb- #14</td>
+<tr>
+<td>9</td>
+<td>Pptb- #15</td>
+<tr>
+<td>10</td>
+<td>Empty</td>
+<tr>
+<td>11</td>
+<td>Empty</td>
+<tr>
+<td>12</td>
+<td>Empty</td>
+<tr>
+<td>13</td>
+<td>Empty</td>
+<tr>
+<td>14</td>
+<td>Empty</td>
+<tr>
+<td>15</td>
+<td>EMpty</td>
+<tr>
+<td>16</td>
+<td>Empty</td>
+<tr>
+<th>Row #2</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<td>1</td>
+<td>Ladder (1kb plus)<td>
+<tr>
+<td>2</td>
+<td>Pptb- #16</td>
+<tr>
+<td>3</td>
+<td>Pptb- #17</td>
+<tr>
+<td>4</td>
+<td>Pptb- #18</td>
+<tr>
+<td>5</td>
+<td>Pptb- #21</td>
+<tr>
+<td>6</td>
+<td>Pptb- #22</td>
+<tr>
+<td>7</td>
+<td>Pptb- #23</td>
+<tr>
+<td>8</td>
+<td>Pptb- #24</td>
+<tr>
+<td>9</td>
+<td>Pptb- #25</td>
+<tr>
+<td>10</td>
+<td>Empty</td>
+<tr>
+<td>11</td>
+<td>Empty</td>
+<tr>
+<td>12</td>
+<td>Empty</td>
+<tr>
+<td>13</td>
+<td>Empty</td>
+<tr>
+<td>14</td>
+<td>Empty</td>
+<tr>
+<td>15</td>
+<td>EMpty</td>
+<tr>
+<td>16</td>
+<td>Empty</td>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/c/c9/17.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">09/6/13</p>
+<div align="center" style="border:1px solid white"><b>Digest of Pptb- and Clos Ori</b></div></br></br>
+<p>3 #17</p>
+<math>500ng X (&#956;L/123.2ng) = 4.06&#956;L</math>
+</br>
+<p>Pptb- #1</p>
+</br>
+<math>500ng X (&#956;L/104.6ng) = 4.78&#956;L</math>
+</br>
+<p>Pptb- #2</p>
+<math>500ng X (&#956;L/123.1ng) = 4.06&#956;L</math>
+</br>
+<p>Pptb- #3</p>
+<math>500ng X (&#956;L/92.7ng) = 5,39&#956;L</math>
+</br>
+<p>Pptb- #4</p>
+<math>500ng X (&#956;L/74.5ng) = 6.71&#956;L</math>
+</br>
+<p>Pptb- #5</p>
+<math>500ng X (&#956;L/114.6ng) = 4.36&#956;L</math>
+</br>
+<p>Pptb- #7</p>
+<math>500ng X (&#956;L/154.8ng) = 3.23&#956;L</math>
+</br>
+<p>Pptb- #14</p>
+<math>500ng X (&#956;L/127.9ng) = 3.91&#956;L</math>
+</br>
+<p>Pptb- #15</p>
+<math>500ng X (&#956;L/110.4ng) = 4.53&#956;L</math>
+</br>
+<p>Pptb- #16</p>
+<math>500ng X (&#956;L/112.4ng) = 4.45&#956;L</math>
+<p>Pptb- #17</p>
+<math>500ng X (&#956;L/124.5ng) = 4.01&#956;L</math>
+</br>
+<p>Pptb- #18</p>
+<math>500ng X (&#956;L/219.8ng) = 2.27&#956;L</math>
+</br>
+<p>Pptb- #21</p>
+<math>500ng X (&#956;L/157.6ng) = 3.17&#956;L</math>
+</br>
+<p>Pptb- #22</p>
+<math>500ng X (&#956;L/145.6ng) = 3.43&#956;L</math>
+</br>
+<p>Pptb- #23</p>
+<math>500ng X (&#956;L/134.2ng) = 3.73&#956;L</math>
+</br>
+<p>Pptb- #24</p>
+<math>500ng X (&#956;L/114.6ng) = 4.36&#956;L</math>
+</br>
+<p>Pptb- #25</p>
+<math>500ng X (&#956;L/189.8ng) = 2.63&#956;L</math>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Sample</th>
+<th>EcoR1</th>
+<th>Pst1</th>
+<th>10x Buffer</th>
+<th>DNA</th>
+<th>PCR dH<sub>2</sub>O</th>
+<th>Total</th>
+<tr>
+<td>Pptb- #1</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.78&#956;L</td>
+<td>9.22&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #2</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.06&#956;L</td>
+<td>9.94&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #3</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>5.39&#956;L</td>
+<td>8.61&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #4</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>6.71&#956;L</td>
+<td>7.29&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #5</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.36&#956;L</td>
+<td>9.64&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #7</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>3.23&#956;L</td>
+<td>10.77&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #14</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>3.91&#956;L</td>
+<td>10.09&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #815</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.53&#956;L</td>
+<td>9.47&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #16</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.45&#956;L</td>
+<td>9.55&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #17</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.02&#956;L</td>
+<td>9.98&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #18</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2.27&#956;L</td>
+<td>11.73&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #21</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>3.17&#956;L</td>
+<td>10.83&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #22</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>3.43&#956;L</td>
+<td>10.57&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #23</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>3.73&#956;L</td>
+<td>10.27&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #24</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.36&#956;L</td>
+<td>9.64&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Pptb- #25</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2.63&#956;L</td>
+<td>11.37&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+</table>
+</br>
+<p>Heat shock for 45 minutes in water bath and 15 minutes at 80&#176;C on heat block to inactivate EcoR1</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">09/6/13</p>
+<div align="center" style="border:1px solid white"><b>Quantification and DNA Extraction of 3A</b></div></br></br>
+<p>QIAprep Spin Miniprep Kit (250)
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/1/1d/86.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<p align="center">
+<img src="<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/0/06/87.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">09/5/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of Plep #1</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row 1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder (1kb plus)</th>
+<tr>
+<th>2</th>
+<th>Plep #1</th>
+<tr>
+<th>3</th>
+<th>Empty</th>
+<tr>
+<th>4</th>
+<th>Empty</th>
+<tr>
+<th>5</th>
+<th>Empty</th>
+<tr>
+<th>6</th>
+<th>Empty</th>
+<tr>
+<th>7</th>
+<th>Empty</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/6/60/85.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">09/5/13</p>
+<div align="center" style="border:1px solid white"><b>Digest of Plep #1</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Amount</th>
+<th>Component</th>
+<tr>
+<td>6.67&#956;L</td>
+<td>Plep</td>
+<tr>
+<td>2&#956;L</td>
+<td>10x Buffer</td>
+<tr>
+<td>2&#956;L</td>
+<td>EcoR1</td>
+<tr>
+<td>2&#956;L</td>
+<td>Pst1</td>
+<tr>
+<td>7.33&#956;L</td>
+<td>PCR Water</td>
+<tr>
+</table>
+</br>
+<math>500ng X (&#956;L/75ng) = 6.67&#956;L</math>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">09/5/13</p>
+<div align="center" style="border:1px solid white"><b>Quantification and DNA Extraction of 3A</b></div></br></br>
+<p>QIAprep Spin Miniprep Kit (250)
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/d/dc/82.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/1/16/83.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/8/82/84.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">09/4/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of Colony PCR</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row #1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<td>1</td>
+<td>Ladder (1kb plus)<td>
+<tr>
+<td>2</td>
+<td>T2 #1</td>
+<tr>
+<td>3</td>
+<td>T2 #2</td>
+<tr>
+<td>4</td>
+<td>T2 #3</td>
+<tr>
+<td>5</td>
+<td>T2 #4</td>
+<tr>
+<td>6</td>
+<td>Ter #5</td>
+<tr>
+<td>7</td>
+<td>T2 #16</td>
+<tr>
+<td>8</td>
+<td>T2 #17</td>
+<tr>
+<td>9</td>
+<td>T2 #18</td>
+<tr>
+<td>10</td>
+<td>T2 #19</td>
+<tr>
+<td>11</td>
+<td>T2 #20</td>
+<tr>
+<td>12</td>
+<td>Empty</td>
+<tr>
+<td>13</td>
+<td>Empty</td>
+<tr>
+<td>14</td>
+<td>Empty</td>
+<tr>
+<td>15</td>
+<td>EMpty</td>
+<tr>
+<td>16</td>
+<td>Empty</td>
+<tr>
+<th>Row #2</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<td>1</td>
+<td>Ladder (1kb plus)<td>
+<tr>
+<td>2</td>
+<td>Pveg2 #1</td>
+<tr>
+<td>3</td>
+<td>Pveg2 #2</td>
+<tr>
+<td>4</td>
+<td>Pveg2 #3</td>
+<tr>
+<td>5</td>
+<td>Pveg2 #4</td>
+<tr>
+<td>6</td>
+<td>Pveg2 #5</td>
+<tr>
+<td>7</td>
+<td>Pveg2 #16</td>
+<tr>
+<td>8</td>
+<td>Pveg2 #17</td>
+<tr>
+<td>9</td>
+<td>Pveg2 #18</td>
+<tr>
+<td>10</td>
+<td>Pveg2 #19</td>
+<tr>
+<td>11</td>
+<td>Pveg2 #20</td>
+<tr>
+<td>12</td>
+<td>Empty</td>
+<tr>
+<td>13</td>
+<td>Empty</td>
+<tr>
+<td>14</td>
+<td>Empty</td>
+<tr>
+<td>15</td>
+<td>EMpty</td>
+<tr>
+<td>16</td>
+<td>Empty</td>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/e/ec/81.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row #1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<td>1</td>
+<td>Ladder (1kb plus)<td>
+<tr>
+<td>2</td>
+<td>Pptb- #1</td>
+<tr>
+<td>3</td>
+<td>Pptb- #2</td>
+<tr>
+<td>4</td>
+<td>Pptb- #3</td>
+<tr>
+<td>5</td>
+<td>Pptb- #4</td>
+<tr>
+<td>6</td>
+<td>Pptb- #5</td>
+<tr>
+<td>7</td>
+<td>Pptb- #16</td>
+<tr>
+<td>8</td>
+<td>Pptb- #17</td>
+<tr>
+<td>9</td>
+<td>Pptb- #18</td>
+<tr>
+<td>10</td>
+<td>Pptb- #19</td>
+<tr>
+<td>11</td>
+<td>Pptb- #20</td>
+<tr>
+<td>12</td>
+<td>Empty</td>
+<tr>
+<td>13</td>
+<td>Empty</td>
+<tr>
+<td>14</td>
+<td>Empty</td>
+<tr>
+<td>15</td>
+<td>EMpty</td>
+<tr>
+<td>16</td>
+<td>Empty</td>
+<tr>
+<th>Row #2</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<td>1</td>
+<td>Ladder (1kb plus)<td>
+<tr>
+<td>2</td>
+<td>Plep #1</td>
+<tr>
+<td>3</td>
+<td>Plep #2</td>
+<tr>
+<td>4</td>
+<td>Plep #3</td>
+<tr>
+<td>5</td>
+<td>Plep #4</td>
+<tr>
+<td>6</td>
+<td>Plep #5</td>
+<tr>
+<td>7</td>
+<td>Plep #16</td>
+<tr>
+<td>8</td>
+<td>Plep #17</td>
+<tr>
+<td>9</td>
+<td>Plep #18</td>
+<tr>
+<td>10</td>
+<td>Plep #19</td>
+<tr>
+<td>11</td>
+<td>Plep #20</td>
+<tr>
+<td>12</td>
+<td>Empty</td>
+<tr>
+<td>13</td>
+<td>Empty</td>
+<tr>
+<td>14</td>
+<td>Empty</td>
+<tr>
+<td>15</td>
+<td>EMpty</td>
+<tr>
+<td>16</td>
+<td>Empty</td>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/f/f7/80.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">09/1/13</p>
+<div align="center" style="border:1px solid white"><b>3A Ligation</b></div></br></br>
+<p>1) Clos Ori +Ptb- + pSB1A3</p>
+<p>1) Clos Ori +Pveg1 + pSB1A3</p>
+<p>1) Clos Ori +Pveg2 + pSB1A3</p>
+<p>1) Clos Ori +Pliag + pSB1A3</p>
+<p>1) mlSR + Plep + pSB1A3</p>
+<p>1) mlSR +Terminator2 (4P) + pSB1A3</p>
+</br>
+<h1></h1>
+<p class = "date">08/30/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of 3A Assembly</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row #1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<td>1</td>
+<td>2 #8<td>
+<tr>
+<td>2</td>
+<td>2 #10</td>
+<tr>
+<td>3</td>
+<td>2 #12</td>
+<tr>
+<td>4</td>
+<td>3 #15</td>
+<tr>
+<td>5</td>
+<td>3 #17</td>
+<tr>
+<td>6</td>
+<td>4 #1</td>
+<tr>
+<td>7</td>
+<td>4 #3</td>
+<tr>
+<td>8</td>
+<td>5 #1</td>
+<tr>
+<td>9</td>
+<td>6 #1</td>
+<tr>
+<td>10</td>
+<td>6 #3</td>
+<tr>
+<td>11</td>
+<td>Ladder (1kb plus)</td>
+<tr>
+<td>12</td>
+<td>Empty</td>
+<tr>
+<td>13</td>
+<td>Empty</td>
+<tr>
+<td>14</td>
+<td>Empty</td>
+<tr>
+<td>15</td>
+<td>EMpty</td>
+<tr>
+<td>16</td>
+<td>Empty</td>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/7/74/74.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/30/13</p>
+<div align="center" style="border:1px solid white"><b>Digest of 3A Assembly</b></div></br></br>
+<p>3 #17</p>
+<math>500ng X (&#956;L/123.2ng) = 4.06&#956;L</math>
+</br>
+<p>6 #1</p>
+</br>
+<math>500ng X (&#956;L/98.7ng) = 5.07&#956;L</math>
+</br>
+<p>6 #3</p>
+<math>500ng X (&#956;L/110.2ng) = 4.54&#956;L</math>
+</br>
+<p>5 #1</p>
+<math>500ng X (&#956;L/162.6ng) = 3.08&#956;L</math>
+</br>
+<p>2 #12</p>
+<math>500ng X (&#956;L/78.8ng) = 6.35&#956;L</math>
+</br>
+<p>3 #15</p>
+<math>500ng X (&#956;L/131.8ng) = 3.79&#956;L</math>
+</br>
+<p>4 #1</p>
+<math>500ng X (&#956;L/146.5ng) = 3.41&#956;L</math>
+</br>
+<p>2 #8</p>
+<math>500ng X (&#956;L/117.7ng) = 4.25&#956;L</math>
+</br>
+<p>2 #10</p>
+<math>500ng X (&#956;L/87.3ng) = 5.73&#956;L</math>
+</br>
+<p>4 #3</p>
+<math>500ng X (&#956;L/82.9ng) = 6.03&#956;L</math>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Sample</th>
+<th>EcoR1</th>
+<th>Pst1</th>
+<th>10x Buffer</th>
+<th>DNA</th>
+<th>PCR dH<sub>2</sub>O</th>
+<th>Total</th>
+<tr>
+<td>3 #17</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.06&#956;L</td>
+<td>9.94&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>6 #1</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>5.07&#956;L</td>
+<td>8.93&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>6 #3</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.54&#956;L</td>
+<td>9.46&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>5 #1</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>3.08&#956;L</td>
+<td>10.92&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>2 #12</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>6.35&#956;L</td>
+<td>7.65&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>3 #15</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>3.79&#956;L</td>
+<td>10.21&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>4 #1</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>3.41&#956;L</td>
+<td>10.59&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>2 #8</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.25&#956;L</td>
+<td>9.75&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>2 #10</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>5.73&#956;L</td>
+<td>8.27&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>4 #3</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>6.03&#956;L</td>
+<td>7.97&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+</table>
+</br>
+<p>Heat shock for 45 minutes in water bath and 15 minutes at 80&#176;C on heat block to inactivate EcoR1</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/30/13</p>
+<div align="center" style="border:1px solid white"><b>Quantification and DNA Extraction of 3A</b></div></br></br>
+<p>QIAprep Spin Miniprep Kit (250)
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/5/55/68.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/1/1f/69.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/27/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of 3A Assembly </b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row #1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<td>1</td>
+<td>Ladder (1kb plus)<td>
+<tr>
+<td>2</td>
+<td>1 #9</td>
+<tr>
+<td>3</td>
+<td>2 #22</td>
+<tr>
+<td>4</td>
+<td>2 #24</td>
+<tr>
+<td>5</td>
+<td>2 #26</td>
+<tr>
+<td>6</td>
+<td>3 #3</td>
+<tr>
+<td>7</td>
+<td>4 #2</td>
+<tr>
+<td>8</td>
+<td>5 #3</td>
+<tr>
+<td>9</td>
+<td>5 #11</td>
+<tr>
+<td>10</td>
+<td>6 #2</td>
+<tr>
+<td>11</td>
+<td>6 #19</td>
+<tr>
+<td>12</td>
+<td>6 #24</td>
+<tr>
+<td>13</td>
+<td>Empty</td>
+<tr>
+<td>14</td>
+<td>Empty</td>
+<tr>
+<td>15</td>
+<td>EMpty</td>
+<tr>
+<td>16</td>
+<td>Empty</td>
+<tr>
+</table>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/26/13</p>
+<div align="center" style="border:1px solid white"><b>Digest of Ter and Adh6</b></div></br></br>
+<p>1 #9</p>
+<math>500ng X (&#956;L/124.7ng) = 4&#956;L</math>
+</br>
+<p>ter #18</p>
+</br>
+<math>500ng X (&#956;L/118.9ng) = 4.21&#956;L</math>
+</br>
+<p>2 #22</p>
+<math>500ng X (&#956;L/76.1ng) = 6.57&#956;L</math>
+</br>
+<p>2 #24</p>
+<math>500ng X (&#956;L/96.4ng) = 5.19&#956;L</math>
+</br>
+<p>2 #26</p>
+<math>500ng X (&#956;L/99.4ng) = 5.03&#956;L</math>
+</br>
+<p>3 #3</p>
+<math>500ng X (&#956;L/101ng) = 4.95&#956;L</math>
+</br>
+<p>4 #2</p>
+<math>500ng X (&#956;L/104ng) = 4.81&#956;L</math>
+</br>
+<p>5 #3</p>
+<math>500ng X (&#956;L/84.6ng) = 5.91&#956;L</math>
+</br>
+<p>5 #11</p>
+<math>500ng X (&#956;L/124.6ng) = 4.01&#956;L</math>
+</br>
+<p>6 #2</p>
+<math>500ng X (&#956;L/78.8ng) = 6.35&#956;L</math>
+</br>
+<p>6 #19</p>
+<math>500ng X (&#956;L/141.4ng) = 3.54&#956;L</math>
+</br>
+<p>6 #24</p>
+<math>500ng X (&#956;L/100.2ng) = 4.99&#956;L</math>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Sample</th>
+<th>EcoR1</th>
+<th>Pst1</th>
+<th>10x Buffer</th>
+<th>DNA</th>
+<th>PCR dH<sub>2</sub>O</th>
+<th>Total</th>
+<tr>
+<td>1 #9</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.21&#956;L</td>
+<td>9.79&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>2 #22</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>6.57&#956;L</td>
+<td>7.43&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>2 #24</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>5.19&#956;L</td>
+<td>8.81&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>2 #26</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>5.03&#956;L</td>
+<td>8.97&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>3 #3</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.95&#956;L</td>
+<td>9.05&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>4 #2</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.81&#956;L</td>
+<td>9.19&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>5 #3</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>5.91&#956;L</td>
+<td>8.09&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>5 #11</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.01&#956;L</td>
+<td>9.99&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>6 #2</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>6.35&#956;L</td>
+<td>7.65&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>6 #19</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>3.54&#956;L</td>
+<td>10.46&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>6 #24</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.99&#956;L</td>
+<td>9.01&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+</table>
+</br>
+<a href = "#top">Back to top</a>
+h1></h1>
+<p class = "date">08/27/13</p>
+<div align="center" style="border:1px solid white"><b>Quantification of 3A Assembly</b></div></br></br>
+<p>QUIAPREP/Spin Miniprep Kit < (250)</p>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/9/99/66.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/26/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of ter and Adh6</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row 1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder (1kb plus)</th>
+<tr>
+<th>2</th>
+<th>ter #6</th>
+<tr>
+<th>3</th>
+<th>ter #18</th>
+<tr>
+<th>4</th>
+<th>ter #24</th>
+<tr>
+<th>5</th>
+<th>Adh6 #8</th>
+<tr>
+<th>6</th>
+<th>Adh6 #14</th>
+<tr>
+<th>7</th>
+<th>Adh6 #15</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/c/c9/17.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/26/13</p>
+<div align="center" style="border:1px solid white"><b>Digest of Ter and Adh6</b></div></br></br>
+<p>ter #6</p>
+<math>500ng X (&#956;L/124.7ng) = 4&#956;L</math>
+</br>
+<p>ter #18</p>
+</br>
+<math>500ng X (&#956;L/108ng) = 4.63&#956;L</math>
+</br>
+<p>ter #24</p>
+<math>500ng X (&#956;L/315ng) = 1.59&#956;L</math>
+</br>
+<p>Adh6 #8</p>
+<math>500ng X (&#956;L/300.3ng) = 12.41&#956;L</math>
+</br>
+<p>Adh6 #14</p>
+<math>500ng X (&#956;L/95.4ng) = 5.24&#956;L</math>
+</br>
+<p>Adh6 #15</p>
+<math>500ng X (&#956;L/328.3ng) = 1.52&#956;L</math>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Sample</th>
+<th>EcoR1</th>
+<th>Pst1</th>
+<th>10x Buffer</th>
+<th>DNA</th>
+<th>PCR dH<sub>2</sub>O</th>
+<th>Total</th>
+<tr>
+<td>ter #6</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.0&#956;L</td>
+<td>10.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>ter #18</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>4.63&#956;L</td>
+<td>9.37&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>ter #24</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>1.59&#956;L</td>
+<td>12.41&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Adh6 #8</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>1.67&#956;L</td>
+<td>12.33&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Adh6 #14</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>5.24&#956;L</td>
+<td>11.76&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Adh6 #15</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>1.52&#956;L</td>
+<td>12.48&#956;L</td>
+<td>20&#956;L</td>
+</table>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/26/13</p>
+<div align="center" style="border:1px solid white"><b>DNA Extraction of Adh6 and Ter</b></div></br></br>
+<p>QIAprep Spin Miniprep Kit (250)</p>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/c/c9/17.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/22/13</p>
+<div align="center" style="border:1px solid white"><b>Transformation of 3A Assembly</b></div></br></br>
+<ol>
+<li>Transform 4&#956;L of ligation product into 100&#956;L aliquots of competent E. coli cells</li>
+<li>Incubate on ice for 30 minutes</li>
+<li>Heat shock in water bath for 60 seconds</li>
+<li>Incubate on ice for 5 minutes</li>
+<li>Add 600&#956;L of psi broth</li>
+<li>Incubate at 37&#176;C for 2 hours at 200 rpm</li>
+<li>Make necessary dilutions and plate out</li>
+</ol>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/22/13</p>
+<div align="center" style="border:1px solid white"><b>3A Ligation</b></div></br></br>
+<p>1) Clos Ori +Pveg2 (20G) + pSB1A3</p>
+<p>1) Clos Ori +Plep (20E) + pSB1A3</p>
+<p>1) Clos Ori +Pveg1 (23B) + pSB1A3</p>
+<p>1) Clos Ori +Pliag (20A) + pSB1A3</p>
+<p>1) mlSR +Terminator1 (6D) + pSB1A3</p>
+<p>1) mlSR +Terminator2 (4P) + pSB1A3</p>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<th>Amount</th>
+<th>Sample</th>
+<tr>
+<td>2&#956;L</td>
+<td>Upstream Part</td>
+<tr>
+<td>2&#956;L</td>
+<td>Downstream Part</td>
+<tr>
+<td>2&#956;L</td>
+<td>Destination Backbone</td>
+<tr>
+<td>2&#956;L</td>
+<td>10x T4 DNA Ligase Buffer</td>
+<tr>
+<td>0.5&#956;L</td>
+<td>T4 DNA Ligase</td>
+<tr>
+<td>10&#956;L</td>
+<td>PCR Water</td>
+<tr>
+</table>
+</br>
+<p>Incubate at room temperature for 10 minutes, then at 80&#176;C for 20 minutes to inactivate EcoR1</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/15/13</p>
+<div align="center" style="border:1px solid white"><b>      </b></div></br></br>
+<p>Clos Ori</p>
+<math>500ng X (440bp/2000bp) X (&#956;L/190.5ng) = 0.577&#956;L 1:105.7&#956;L</math>
+</br>
+<p>mlSR</p>
+<math>500ng X (750bp/2000bp) X (&#956;L/260.4ng) = 0.908&#956;L 1:109.08&#956;L</math>
+</br>
+<p>Pliag</p>
+<math>500ng X (121bp/2000bp) X (&#956;L/150.1ng) = 0.201&#956;L 1:102.01&#956;L</math>
+</br>
+<p>Pveg1</p>
+<math>500ng X (100bp/2000bp) X (&#956;L/90.0ng) = 0.278&#956;L 1:102.78&#956;L</math>
+</br>
+<p>Plep</p>
+<math>500ng X (157bp/2000bp) X (&#956;L/235.0ng) = 0.167&#956;L 1:101.67&#956;L</math>
+</br>
+<p>Pveg2</p>
+<math>500ng X (237bp/2000bp) X (&#956;L/192.3ng) = 0.308&#956;L 1:103.08&#956;L</math>
+</br>
+<p>Terminator</p>
+<math>500ng X (50bp/2000bp) X (&#956;L/108.6ng) = 0.115&#956;L 1:101.15&#956;L</math>
+</br>
+<p>Terminator</p>
+<math>500ng X (50bp/2000bp) X (&#956;L/131.8ng) = 0.095&#956;L 1:100.95&#956;L</math>
+</br>
+<p>pSB1A3</p>
+<math>500ng X (&#956;L/31.8ng) = 2&#956;L</math>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Sample</th>
+<th>EcoR1</th>
+<th>Pst1</th>
+<th>Xba1</th>
+<th>Spe1</th>
+<th>100x Bovine Serum Albumin</th>
+<th>10x Buffer</th>
+<th>DNA</th>
+<th>PCR dH<sub>2</sub>O</th>
+<th>Total</th>
+<tr>
+<td>Clos Ori</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>-</td>
+<td>-</td>
+<td>1&#956;L</td>
+<td>5&#956;L</td>
+<td>5.7&#956;L</td>
+<td>36.3&#956;L</td>
+<td>50&#956;L</td>
+<tr>
+<td>mlSR</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>-</td>
+<td>-</td>
+<td>1&#956;L</td>
+<td>5&#956;L</td>
+<td>9.08&#956;L</td>
+<td>32.92&#956;L</td>
+<td>50&#956;L</td>
+<tr>
+<td>Pliag</td>
+<td>-</td>
+<td>-</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>5&#956;L</td>
+<td>5.7&#956;L</td>
+<td>39.99&#956;L</td>
+<td>50&#956;L</td>
+<tr>
+<td>Pveg1</td>
+<td>-</td>
+<td>-</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>5&#956;L</td>
+<td>2.78&#956;L</td>
+<td>39.22&#956;L</td>
+<td>50&#956;L</td>
+<tr>
+<td>Pveg2</td>
+<td>-</td>
+<td>-</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>5&#956;L</td>
+<td>1.67&#956;L</td>
+<td>40.33&#956;L</td>
+<td>50&#956;L</td>
+<tr>
+<td>Plep</td>
+<td>-</td>
+<td>-</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>5&#956;L</td>
+<td>3.08&#956;L</td>
+<td>38.92&#956;L</td>
+<td>50&#956;L</td>
+<tr>
+<td>Terminator 6D</td>
+<td>-</td>
+<td>-</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>5&#956;L</td>
+<td>1.15&#956;L</td>
+<td>40.85&#956;L</td>
+<td>50&#956;L</td>
+<tr>
+<td>Temrinator 4P</td>
+<td>-</td>
+<td>-</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>5&#956;L</td>
+<td>0.95&#956;L</td>
+<td>41.05&#956;L</td>
+<td>50&#956;L</td>
+<tr>
+<td>pSB1A3</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>-</td>
+<td>-</td>
+<td>1&#956;L</td>
+<td>5&#956;L</td>
+<td>16&#956;L</td>
+<td>26&#956;L</td>
+<td>50&#956;L</td>
+<tr>
+</table>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/21/13</p>
+<div align="center" style="border:1px solid white"><b>PCR of BktB Fragments 1 & 2 used for SDM Step1      </b></div></br></br>
+<p>Previous attempt produced a lot of extraneous artifacts. </p>
+<p>Primer concentration was too high and so was the template concentrations</p>
+<p>Used Grad program previously used to run PCR machine</p>
+<p>50&#176;C to 60&#176;V used 4 and 0</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/21/13</p>
+<div align="center" style="border:1px solid white"><b>Quantification of Backbones Post-SPRI Based Size Selection </b></div></br></br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/c/c9/17.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<p>The concentrations of pSB1A3 is low. Therefore, the SPRI based size selection was conducted on it once more. The gel results are displayed below.</p>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/3/38/60.1.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/e/e6/60.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/21/13</p>
+<div align="center" style="border:1px solid white"><b>Transformation of Ter + pSB1C3 and Adh6 + pSB1C3</b></div></br></br>
+<ol>
+<li>Thaw competent Top10 cell of E. coli from -80&#176;C (100&#956;L aliquots)</li>
+<li>Add 20&#956;L of ligation product to Top 10 E. coli</li>
+<li>Incubate on ice for 30 minutes</li>
+<li>Heat shock in water bath for exactly 60 seconds</li>
+<li>Incubate on ice for 5 minutes</li>
+<li>Add 600&#956;L of psi broth</li>
+<li>Incubate at 37&#176;C for 2 hours at 200 rpm</li>
+<li>Make dilutions using 90&#956;L of psi broth: 1:10, 1:100, 1:1000</li>
+<li>Plate out 25&#956;L undiluted and diluted solutions onto a petri dish</li>
+</ol>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row 1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder (1kb plus)</th>
+<tr>
+<th>2</th>
+<th>pSB1A3</th>
+<tr>
+<th>3</th>
+<th>Empty</th>
+<tr>
+<th>4</th>
+<th>Empty</th>
+<tr>
+<th>5</th>
+<th>Empty</th>
+<tr>
+<th>6</th>
+<th>Empty</th>
+<tr>
+<th>7</th>
+<th>Empty</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/21/13</p>
+<div align="center" style="border:1px solid white"><b>Gel Post SPRI Based Size Selection</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row 1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder (1kb plus)</th>
+<tr>
+<th>2</th>
+<th>pSB1A3</th>
+<tr>
+<th>3</th>
+<th>pSB1C3</th>
+<tr>
+<th>4</th>
+<th>pSB1K3</th>
+<tr>
+<th>5</th>
+<th>Empty</th>
+<tr>
+<th>6</th>
+<th>Empty</th>
+<tr>
+<th>7</th>
+<th>Empty</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/c/c9/17.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/21/13</p>
+<div align="center" style="border:1px solid white"><b>SPRI Based Size Selection</b></div></br></br>
+<p>Use the instructions labeled “left side size slection.” This kit will hopefully remove the primer dimer from our product. Remove the primer dimer from the backbone PCR clean-up.</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/21/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of Refined Backbone PCR</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row 1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder (1kb plus)</th>
+<tr>
+<th>2</th>
+<th>pSB1A3</th>
+<tr>
+<th>3</th>
+<th>pSB1C3</th>
+<tr>
+<th>4</th>
+<th>pSB1K3</th>
+<tr>
+<th>5</th>
+<th>Empty</th>
+<tr>
+<th>6</th>
+<th>Empty</th>
+<tr>
+<th>7</th>
+<th>Empty</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/c/c9/17.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/21/13</p>
+<div align="center" style="border:1px solid white"><b>PCR of pSB1A3, pSB1K3, pSB1K3 (refined)</b></div></br></br>
+<p>Sample G in the primer matrix will be used as a template for this particular PCR</p>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Label</th>
+<th>Amount</th>
+<th>Component</th>
+<tr>
+<td>PCR # 1</td>
+<td>3&#956;L</td>
+<td>SBprep-3P-1</td>
+<tr>
+<td></td>
+<td>1&#956;L</td>
+<td>SBprep-2Ea</td>
+<tr>
+<td></td>
+<td>2.0&#956;L</td>
+<td>pSB1A3</td>
+<tr>
+<td></td>
+<td>94&#956;L</td>
+<td>PCR Supermix High Fidelity</td>
+<tr>
+<td>PCR #3</td>
+<td>3&#956;L</td>
+<td>SBprep-3P-1</td>
+<tr>
+<td></td>
+<td>1&#956;L</td>
+<td>SBprep-2Ea</td>
+<tr>
+<td></td>
+<td>2.0&#956;L</td>
+<td>pSB1C3</td>
+<tr>
+<td>PCR #2</td>
+<td>3&#956;L</td>
+<td>SBprep-3P-1</td>
+<tr>
+<td></td>
+<td>1&#956;L</td>
+<td>SBprep-2Ea</td>
+<tr>
+<td></td>
+<td>2.0&#956;L</td>
+<td>pSB1K3</td>
+<tr>
+<td></td>
+<td>94&#956;L</td>
+<td>PCR Supermix High Fidelity</td>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/c/c9/17.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/20/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of Fifth PCR of Backbones</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row 1</th>
+<tr>
+<th>Lane Number (1kb plus)</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder</th>
+<tr>
+<th>2</th>
+<th>E</th>
+<tr>
+<th>3</th>
+<th>G</th>
+<tr>
+<th>4</th>
+<th>Empty</th>
+<tr>
+<th>5</th>
+<th>Empty</th>
+<tr>
+<th>6</th>
+<th>Empty</th>
+<tr>
+<th>7</th>
+<th>Empty</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/c/c2/41.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/15/13</p>
+<div align="center" style="border:1px solid white"><b>      </b></div></br></br>
+<p>Use the two concentrations of E and G on 20-August</p>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/c/c9/17.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/20/13</p>
+<div align="center" style="border:1px solid white"><b>PCR of Backbone Discussion</b></div></br></br>
+<p>After discussing the results with Dr. Wawrik, it has been decided to cut the number of cycles down to thirty, and to increase the template used by two.</p>
+<p>The best concentrations of primers to be used are indicated by the results of the primer matrix on letters E and G. F appeared to have been a probable pipetting error.</p>
+<p>List of reactants and amount for primer matrix PCR optimization</p>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Label</th>
+<th>Amount</th>
+<th>Component</th>
+<tr>
+<td>E</td>
+<td>0.5&#956;L</td>
+<td>SBprep-3P-1 (1:2 dilutions) use 1&#956;L </td>
+<tr>
+<td></td>
+<td>1.5&#956;L</td>
+<td>SBprep-2Ea</td>
+<tr>
+<td>1.0&#956;L</td>
+<td>Template</td>
+<tr>
+<td></td>
+<td>46.5&#956;L</td>
+<td>PCR Supermix High Fidelity</td>
+<tr>
+<td>G</td>
+<td>1.5&#956;L</td>
+<td>SBprep-3P-1</td>
+<tr>
+<td></td>
+<td>0.5&#956;L</td>
+<td>SBprep-2Ea (1:2 dilutions) use 1&#956;L </td>
+<tr>
+<td></td>
+<td>1.0&#956;L</td>
+<td>Template</td>
+<tr>
+<td></td>
+<td>46.5&#956;L</td>
+<td>PCR Supermix High Fidelity</td>
+</table>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/19/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of Ter and Adh6 Post Digest</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row 1</th>
+<tr>
+<th>Lane Number (1kb plus)</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder</th>
+<tr>
+<th>2</th>
+<th>Adh6 #19</th>
+<tr>
+<th>3</th>
+<th>Adh6 #3</th>
+<tr>
+<th>4</th>
+<th>Ter #5</th>
+<tr>
+<th>5</th>
+<th>Ter #23</th>
+<tr>
+<th>6</th>
+<th>Ter #26</th>
+<tr>
+<th>7</th>
+<th>Ter #9</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/c/c2/41.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/14/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of pSB1K3, pSB1C3, pSB1A3 Stocks from 9-May </b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row 1</th>
+<tr>
+<th>Lane Number (1kb plus)</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder</th>
+<tr>
+<th>2</th>
+<th>pSB1A3</th>
+<tr>
+<th>3</th>
+<th>pSB1C3</th>
+<tr>
+<th>4</th>
+<th>pSB1K3</th>
+<tr>
+<th>5</th>
+<th>Empty</th>
+<tr>
+<th>6</th>
+<th>Empty</th>
+<tr>
+<th>7</th>
+<th>Empty</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/4/4d/40.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/14/13</p>
+<div align="center" style="border:1px solid white"><b>PCR Clean-up Quanitification      </b></div></br></br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/4/49/39.1.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<p>Although the concentrations are good, the purity is still in question. It is an improvement on the PCR from 13-August</p>
+<p>One possibility is that the original stocks used as templates in the PCR may not be pure enough. Therefore, a gel will be run on these original stocks</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/14/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of pSB1K3, pSB1C3, pSB1A3 Post Clean-up</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row 1</th>
+<tr>
+<th>Lane Number (1kb plus)</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder</th>
+<tr>
+<th>2</th>
+<th>pSB1A3</th>
+<tr>
+<th>3</th>
+<th>pSB1C3</th>
+<tr>
+<th>4</th>
+<th>pSB1K3</th>
+<tr>
+<th>5</th>
+<th>Empty</th>
+<tr>
+<th>6</th>
+<th>Empty</th>
+<tr>
+<th>7</th>
+<th>Empty</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/6/68/39.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/14/13</p>
+<div align="center" style="border:1px solid white"><b>PCR Clean-up of the Three Backbones</b></div></br></br>
+<p>QIAquick PCR purification kit (250) is used before viewing the post clean-up product on a gel</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/14/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of BktB Fragments</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row 1</th>
+<tr>
+<th>Lane Number (1kb plus)</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder</th>
+<tr>
+<th>2</th>
+<th>50&#176;C</th>
+<tr>
+<th>3</th>
+<th> </th>
+<tr>
+<th>4</th>
+<th> </th>
+<tr>
+<th>5</th>
+<th>60&#176;C </th>
+<tr>
+<th>6</th>
+<th>Empty</th>
+<tr>
+<th>7</th>
+<th>Empty</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+</br>
+<p>What happened here? What is in the lanes above the comb?</p>
+<p>Many have some product and we will use the forward and reverse primers to get the whole gene, if present</p>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/8/87/38.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/14/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of pSB1K3, pSB1C3, pSB1A3 Pre Clean-up</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row 1</th>
+<tr>
+<th>Lane Number (1kb plus)</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder</th>
+<tr>
+<th>2</th>
+<th>pSB1A3</th>
+<tr>
+<th>3</th>
+<th>pSB1C3</th>
+<tr>
+<th>4</th>
+<th>pSB1K3</th>
+<tr>
+<th>5</th>
+<th>Empty</th>
+<tr>
+<th>6</th>
+<th>Empty</th>
+<tr>
+<th>7</th>
+<th>Empty</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/3/3f/37.1.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/14/13</p>
+<div align="center" style="border:1px solid white"><b>Quantified Backbone Stocks for PCR</b></div></br></br>
+<p>The following stock are to be used for backbone amplification in the PCR procedure illustrated on 14-August</p>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/8/87/37.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/14/13</p>
+<div align="center" style="border:1px solid white"><b>Preparing an Equimolar solution of BktB Fragments for PCR to Stitch</b></div></br></br>
+<p>Fragment 1: 340bp – 125.6ng/&#956;L</p>
+</br>
+<p>Fragment 1: 660bp – 66.3ng/&#956;L</p>
+</br>
+<p>Fragment 1: 300bp – 231.7ng/&#956;L</p>
+</br>
+<p>Use 1&#956;L of fragment 3 = 231.7ng/&#956;L</p>
+<p>Fragment 2:</p>
+<math>(221.7) X (660/300) = 520ng x (1&#956;L/66.3ng) = 7.7&#956;L </math>
+<p>Fragment 3:</p>
+<math>(221.7) X (340/300) = 263ng x (1&#956;L/125.6ng) = 2.1&#956;L </math>
+</br>
+<p>Added to a 1.5mL epi tube and diluted to 200&#956;L</p>
+<math>1004ng of DNA/ 200&#956;L = 5ng/&#956;L final concentration of DNA</math>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/14/13</p>
+<div align="center" style="border:1px solid white"><b>PCR of i-GEM Plasmid Backbones Continued</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>PCR #1</th>
+<tr>
+<th>Amount (&#956;L)</th>
+<th>Component</th>
+<tr>
+<td>100</td>
+<td>PCR Supermix High Fidelity</tD>
+<tr>
+<td>0.7</td>
+<td>SB-prep-3P-1</td>
+<tr>
+<td>0.7</td>
+<td>SB-prep-2Ea</td>
+<tr>
+<td>0.6</td>
+<td>pSB1A3 (1ng/&#956;L)</td>
+<tr>
+<th>PCR #2</th>
+<tr>
+<th>Amount (&#956;L)</th>
+<th>Component</th>
+<tr>
+<td>100</td>
+<td>PCR Supermix High Fidelity</tD>
+<tr>
+<td>0.7</td>
+<td>SB-prep-3P-1</td>
+<tr>
+<td>0.7</td>
+<td>SB-prep-2Ea</td>
+<tr>
+<td>0.6</td>
+<td>pSB1C3 (1ng/&#956;L)</td>
+<tr>
+<th>PCR #3</th>
+<tr>
+<th>Amount (&#956;L)</th>
+<th>Component</th>
+<tr>
+<td>100</td>
+<td>PCR Supermix High Fidelity</tD>
+<tr>
+<td>0.7</td>
+<td>SB-prep-3P-1</td>
+<tr>
+<td>0.7</td>
+<td>SB-prep-2Ea</td>
+<tr>
+<td>0.6</td>
+<td>pSB1K3 (1ng/&#956;L)</td>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/c/c1/35.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/14/13</p>
+<div align="center" style="border:1px solid white"><b>Adjusted PCR of iGEM Plasmid Backbones</b></div></br></br>
+<p>Diluting concentrations of pSB1A3, pSB1C3, pSB1K3 to 1ng/&#956;L</p>
+<p>Quantification of current backbones to confirm concentrations before conducting dilution</p>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/0/0a/34.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<p>pSB1A3</p>
+<math>Vf = (27.5ng/&#956;L) X (1&#956;L) X (1&#956;L/1ng) = 27.5&#956;L – 1&#956;L of template = 26.5&#956;L of PCR water</math>
+</br>
+<p>pSB1C3</p>
+<math>Vf = (39.9ng/&#956;L) X (1&#956;L) X (1&#956;L/1ng) = 39.9&#956;L – 1&#956;L of template = 38.9&#956;L of PCR water</math>
+</br>
+<p>pSB1K3</p>
+<math>Vf = (29.4ng/&#956;L) X (1&#956;L) X (1&#956;L/1ng) = 29.4&#956;L – 1&#956;L of template = 28.4&#956;L of PCR water</math>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/13/13</p>
+<div align="center" style="border:1px solid white"><b>Quantification of PCR Clean-up</b></div></br></br>
+<p>The concentrations are good, however the purity of the product is not. Actions taken to remedy this problem are to run another PCR using 1ng/&#956;L of template and running the third step of the PCR cycle at 68&#176;C for 5 minutes.</p>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/9/9c/33.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/13/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of pSB1K3, pSB1C3, pSB1A3 Post Clean-up</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row 1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder</th>
+<tr>
+<th>2</th>
+<th>pSB1A3</th>
+<tr>
+<th>3</th>
+<th>pSB1C3</th>
+<tr>
+<th>4</th>
+<th>pSB1K3</th>
+<tr>
+<th>5</th>
+<th>Empty</th>
+<tr>
+<th>6</th>
+<th>Empty</th>
+<tr>
+<th>7</th>
+<th>Empty</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/c/ce/32.1.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/13/13</p>
+<div align="center" style="border:1px solid white"><b>PCR Clean-up of pSB1K3, pSB1C3, pSB1A3</b></div></br></br>
+<p>The three backbones were cleaned up using QIAquick PCR Purification Kit (250) so that the next gel will much more clearly display the backbone.</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/13/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of pSB1K3, pSB1C3, pSB1A3</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row 1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder</th>
+<tr>
+<th>2</th>
+<th>pSB1K3</th>
+<tr>
+<th>3</th>
+<th>pSB1C3</th>
+<tr>
+<th>4</th>
+<th>pSB1A3</th>
+<tr>
+<th>5</th>
+<th>Empty</th>
+<tr>
+<th>6</th>
+<th>Empty</th>
+<tr>
+<th>7</th>
+<th>Empty</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/1/16/32.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+h1></h1>
+<p class = "date">08/13/13</p>
+<div align="center" style="border:1px solid white"><b>Plasmid Extraction of AaDc-, AaDc+, Thl+, Thl-, and Ptb+</b></div></br></br>
+<p>QUIAPREP/Spin Miniprep Kit < (250)</p>
+<p>Cells were centrifuged from an inoculated broth</p>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/1/19/30.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/6/68/30.1.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<p>None of the concentrations obtained are high enough to work with. Must go about finding a more efficient plasmid extraction for these samples.</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/13/13</p>
+<div align="center" style="border:1px solid white"><b>PCR of i-GEM Plasmid Backbones</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>PCR #1</th>
+<tr>
+<th>Amount (&#956;L)</th>
+<th>Component</th>
+<tr>
+<td>100</td>
+<td>PCR Supermix High Fidelity</tD>
+<tr>
+<td>0.7</td>
+<td>SB-prep-3P-1</td>
+<tr>
+<td>0.7</td>
+<td>SB-prep-2Ea</td>
+<tr>
+<td>0.6</td>
+<td>pSB1C3 (43ng/&#956;L)</td>
+<tr>
+<th>PCR #2</th>
+<tr>
+<th>Amount (&#956;L)</th>
+<th>Component</th>
+<tr>
+<td>100</td>
+<td>PCR Supermix High Fidelity</tD>
+<tr>
+<td>0.7</td>
+<td>SB-prep-3P-1</td>
+<tr>
+<td>0.7</td>
+<td>SB-prep-2Ea</td>
+<tr>
+<td>0.6</td>
+<td>pSB1C3 (74.8ng/&#956;L)</td>
+<tr>
+<th>PCR #3</th>
+<tr>
+<th>Amount (&#956;L)</th>
+<th>Component</th>
+<tr>
+<td>100</td>
+<td>PCR Supermix High Fidelity</tD>
+<tr>
+<td>0.7</td>
+<td>SB-prep-3P-1</td>
+<tr>
+<td>0.7</td>
+<td>SB-prep-2Ea</td>
+<tr>
+<td>0.6</td>
+<td>pSB1C3 (55.1ng/&#956;L)</td>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/5/50/29.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/14/13</p>
+<div align="center" style="border:1px solid white"><b>Post-Cleanup of BktB PCR Products</b></div></br></br>
+<p>All Fragment 1’s, lanes 8 and 9 of Fragment 2, and all Fragment</p>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/2/22/28.1.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/13/13</p>
+<div align="center" style="border:1px solid white"><b>PCR of BktB from PCR Tempalte</b></div></br></br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/b/b9/28.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/9/13</p>
+<div align="center" style="border:1px solid white"><b>Gel of BktB Fragments</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Row #1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<th>Temperature (&#176;C)</th>
+<tr>
+<td>1</td>
+<td>Ladder<td>
+<tr>
+<td>2</td>
+<td>Fragment 1</td>
+<td>50</td>
+<tr>
+<td>3</td>
+<td>Fragment 1</td>
+<tr>
+<td>4</td>
+<td>Fragment 1</td>
+<tr>
+<td>5</td>
+<td>Fragment 1</td>
+<td>60</td>
+<tr>
+<td>6</td>
+<td>Fragment 2</td>
+<td>50</td>
+<tr>
+<td>7</td>
+<td>Fragment 2</td>
+<tr>
+<td>8</td>
+<td>Fragment 2</td>
+<tr>
+<td>9</td>
+<td>Fragment 2</td>
+<td>60</td>
+<tr>
+<td>10</td>
+<td>Fragment 3</td>
+<td>50</td>
+<tr>
+<td>11</td>
+<td>Fragment 3</td>
+<tr>
+<td>12</td>
+<td>Fragment 3</td>
+<tr>
+<td>13</td>
+<td>Fragment 3</td>
+<td>60</td>
+<tr>
+<td>14</td>
+<td>Empty</td>
+<tr>
+<td>15</td>
+<td>EMpty</td>
+<tr>
+<td>16</td>
+<td>Empty</td>
+<tr>
+</table>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/f/ff/27.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<p>Based on 2% agarose gel, 115V for about 30 minutes</p>
+<p>Going to PCR the whole gene to see if I can reduce non-specific products in fragment #2</p>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/7/79/27.1.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/8/13</p>
+<div align="center" style="border:1px solid white"><b>PCR of BktB Fragments</b></div></br></br>
+<p>Preformed gradient PCR for all 3 BktB fragments</p>
+<p>Undiluted template was used</p>
+</br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/2/25/26.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/7/13</p>
+<div align="center" style="border:1px solid white"><b>Transformation of Top10 with ter in pSB1C3</b></div></br></br>
+<ol>
+<li>Thaw competent 100&#956;L aliquot of E. coli cells from -80&#176;C freezer</li>
+<li>Add 20&#956;L of ligation poduct to Top10 E. coli</li>
+<li>Incubate on ice for 30 minutes</li>
+<li>Heat shock in water bath for 60 seconds</li>
+<li>Incubate on ice for 5 minutes</li>
+<li>Add 600&#956;L of psi broth</li>
+<li>Incubate at 37&#176;C for 2 hours at 200 rpm</li>
+<li>Make a dilution series of 1:10, 1:100, 1:1000, and 1:10000</li>
+</ol>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/7/13</p>
+<div align="center" style="border:1px solid white"><b>Ligation of pSB1C3 and Adh6</b></div></br></br>
+<p>Into 1.5mL microcentrifuge tubes</p>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Amount (&#956;L)</th>
+<th>Component</th>
+<tr>
+<td>20</td>
+<td>Heat killed digest of pSB1C3</td>
+<tr>
+<td>20</td>
+<td>Heat killed digest of Adh6</td>
+<tr>
+<td>1.0</td>
+<td>T4 Ligase</td>
+<tr>
+<td>5.0</td>
+<td>10X T4 Ligase Buffer</td>
+<tr>
+<td>4.0</td>
+<td>Nuclease Free PCR Water</td>
+<tr>
+<th>50</th>
+<th>Total</th>
+<tr>
+</table>
+</br>
+<p>Incubate on bench for 45 minutes</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/9/13</p>
+<div align="center" style="border:1px solid white"><b>digestion of ter</b></div></br></br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Sample</th>
+<th>EcoR1</th>
+<th>Pst1</th>
+<th>10x Buffer</th>
+<th>DNA</th>
+<th>PCR dH<sub>2</sub>O</th>
+<th>Total</th>
+<tr>
+<td>ter #23</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>2.5&#956;L</td>
+<td>14.5&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>ter #7</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1.5&#956;L</td>
+<td>15.5&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>ter #26</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>2&#956;L</td>
+<td>15&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>ter #5</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>2&#956;L</td>
+<td>15&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>pSB1C3</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>2.5&#956;L</td>
+<td>14.5&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+</table>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/9/13</p>
+<div align="center" style="border:1px solid white"><b>Plasmid Extraction of AaDc- and Ptb-</b></div></br></br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/7/71/21.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/7/13</p>
+<div align="center" style="border:1px solid white"><b>Restriction Digest of Ccell Adh1 and Adh2</b></div></br></br>
+<p>Adh1 #5</p>
+<math>500ng X (&#956;L/297.2ng) = 1.68&#956;L</math>
+</br>
+<p>Adh1 #19</p>
+<math>500ng X (&#956;L/89.3ng) = 5.60&#956;L</math>
+</br>
+<p>Adh1 #24</p>
+<math>500ng X (&#956;L/261.6ng) = 1.91&#956;L</math>
+</br>
+<p>Adh1 #4</p>
+<math>500ng X (&#956;L/264.0ng) = 1.89&#956;L</math>
+</br>
+<p>Adh1 #13</p>
+<math>500ng X (&#956;L/588.6ng) = 0.85&#956;L</math>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Sample</th>
+<th>EcoR1</th>
+<th>Pst1</th>
+<th>10x Buffer</th>
+<th>DNA</th>
+<th>PCR dH<sub>2</sub>O</th>
+<th>Total</th>
+<tr>
+<td>Adh1 #5</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>1.5&#956;L</td>
+<td>12.5&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Adh1 #19</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>5.5&#956;L</td>
+<td>8.5&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Adh1 #24</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>12&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Adh1 #4</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>12&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Adh1 #13</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>2&#956;L</td>
+<td>1&#956;L</td>
+<td>13&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+</table>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Sample</th>
+<th>EcoR1</th>
+<th>Pst1</th>
+<th>10x Buffer</th>
+<th>DNA</th>
+<th>PCR dH<sub>2</sub>O</th>
+<th>Total</th>
+<tr>
+<td>Adh6 #5</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>5.0&#956;L</td>
+<td>12.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Adh6 #19</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>2.0&#956;L</td>
+<td>15.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Adh6 #3</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>3.0&#956;L</td>
+<td>14.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>Adh6 #15</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>3.0&#956;L</td>
+<td>14.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>pSB1C3</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>2.5&#956;L</td>
+<td>14.5&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+</table>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/7/13</p>
+<div align="center" style="border:1px solid white"><b>Gel for Post-Digest of Promoters and Terminators</b></div></br></br>
+<p>Using 2% agarose in the gel</p>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Gel 1</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder</th>
+<tr>
+<th>2</th>
+<th>#16-6D</th>
+<tr>
+<th>3</th>
+<th>#16-23B</th>
+<tr>
+<th>4</th>
+<th>#9-4P</th>
+<tr>
+<th>5</th>
+<th>#9-6D</th>
+<tr>
+<th>6</th>
+<th>#3-23B</th>
+<tr>
+<th>7</th>
+<th>#14-20E</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+<p align="center">
+<img src=" https://static.igem.org/mediawiki/2013/8/88/18.png
+" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Gel 2</th>
+<tr>
+<th>Lane Number</th>
+<th>Sample</th>
+<tr>
+<th>1</th>
+<th>Ladder</th>
+<tr>
+<th>2</th>
+<th>#15-20A</th>
+<tr>
+<th>3</th>
+<th>#7-20G</th>
+<tr>
+<th>4</th>
+<th>#7-4D</th>
+<tr>
+<th>5</th>
+<th>#14-20A</th>
+<tr>
+<th>6</th>
+<th>#10-4P</th>
+<tr>
+<th>7</th>
+<th>#11-20E</th>
+<tr>
+<th>8</th>
+<th>Empty</th>
+<tr>
+</table>
+<p align="center">
+<img src=" https://static.igem.org/mediawiki/2013/4/40/18.1.png
+" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<p>Samples display a possible insert, and will be sent off for sequencing</p>
+<p>3-23B, 16-23B, 14-20E, 15-20A, 16-6D, 14-20A, and 10-4P</p>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/7/13</p>
+<div align="center" style="border:1px solid white"><b>Modified Restriction Digest of Promoters and Terminators</b></div></br></br>
+<p align="center">
+<img src=" https://static.igem.org/mediawiki/2013/c/c9/17.png
+" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
+<tr>
+<th>Sample</th>
+<th>EcoR1</th>
+<th>Pst1</th>
+<th>10x Buffer</th>
+<th>DNA</th>
+<th>PCR dH<sub>2</sub>O</th>
+<th>Total</th>
+<tr>
+<td>#16-6D</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>10.0&#956;L</td>
+<td>7.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>#16-23B</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>10.0&#956;L</td>
+<td>7.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>#9-4P</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>10.0&#956;L</td>
+<td>7.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>#9-6D</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>10.0&#956;L</td>
+<td>7.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>#3-23B</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>10.0&#956;L</td>
+<td>7.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>#14-20E</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>10.0&#956;L</td>
+<td>7.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>#15-20A</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>10.0&#956;L</td>
+<td>7.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>#7-20G</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>10.0&#956;L</td>
+<td>7.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>#7-4D</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>10.0&#956;L</td>
+<td>7.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>#14-20A</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>10.0&#956;L</td>
+<td>7.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>#10-4P</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>10.0&#956;L</td>
+<td>7.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+<td>#11-20E</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>1&#956;L</td>
+<td>10.0&#956;L</td>
+<td>7.0&#956;L</td>
+<td>20&#956;L</td>
+<tr>
+</table>
+</br>
+<a href = "#top">Back to top</a>
+<h1></h1>
+<p class = "date">08/7/13</p>
+<div align="center" style="border:1px solid white"><b>Plasmid Extraction of Ccell of Adh1 and Adh2</b></div></br></br>
+<p>QIAPrep Spin Miniprep Kit (250)</p>
+</br>
+<p>The gene’s origin is unknown, and will be found upon sequencing</p>
+</br>
+<p align="center">
+<img src=" https://static.igem.org/mediawiki/2013/c/c9/17.png
+" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
+<a href = "#top">Back to top</a>
 <h1></h1>
@@ Line 2,111: / Line 5,291: @@
 <p class = "date">07/22/13</p>
 <div align="center" style="border:1px solid white"><b>PCR Cleanup of Cs Adh PCR Products </b></div></br></br>
-----------------------------pic---------------------------------
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/0/03/99.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
 </br>
 <a href = "#top">Back to top</a>
@@ Line 2,122: / Line 5,305: @@
 <p>Extracted using Qiagen plasmid prep kit</p>
 </br>
-------------------------pic----------------------------------------
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/e/ec/98.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
 </br>
 <a href = "#top">Back to top</a>
@@ Line 2,133: / Line 5,319: @@
 <p>Preformed PCR to amplify Adh out of C. cellulolyticum</p>
 </br>
----------------------------pic---------------------------------------
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/f/fc/97.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
 </br>
 <a href = "#top">Back to top</a>
@@ Line 2,143: / Line 5,332: @@
 <p>Choose lower temperature (50&#176;C) for annealing.</p>
 </br>
--------------------------pic--------------------------------------
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/e/eb/96.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
 </br>
 <p>Still have primer dimer, or primers may be sitting down at another place on the genomic DNA</p>
@@ Line 2,155: / Line 5,348: @@
 <p>No colonies have grown on the plates incubated with transformed Top 10 cells with pSB1C3 and various promoters plated on July 16 2013. We did a nanodrop quantification of the ligationproducts.</p>
 </br>
------------------------PIC----------------------------------------------------
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/c/c9/95.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
 </br>
 <p>While the 260/280 ratios seem high, Dr. wawrick says that they aren’t troubling. However, the amount of DNA quantified is unrealistic. Since, we only started with 100ng of insert and 200ng of vector. We ran a gel to see if what we quantified is DNA and not RNA.</p>
@@ Line 2,408: / Line 5,604: @@
 <p class = "date">07/18/13</p>
 <div align="center" style="border:1px solid white"><b>Plasmid Prep of Transformed Top10 Cells with pSB1C3 + mlsR and puc19 + KIVD</b></div></br></br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/7/77/89.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
 <a href = "#top">Back to top</a>
 <h1></h1>
 <p class = "date">07/17/13</p>
 <div align="center" style="border:1px solid white"><b>DNA Extraction from Ca, Cs, Cb, and Cc using MoBio Power Soil DNA Extraction Kit </b></div></br></br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2013/9/9d/88.png" alt="HTML tutorial" width="300" height="300" ></a></p>
+</p>
+</br>
 <a href = "#top">Back to top</a>
-<h1></h1>
-<p class = "date">05/16/13</p>
-<p>???????????????</p>
-</br>
-<a href = "#top">Back to top</a>
--->
-<h1></h1>
-<p class = "date">07/11/13</p>
-<div align="center" style="border:1px solid white"><b>Dual Digest of pSB1C3 and mlsR Part </b></div></br></br>
-<a href = "#top">Back to top</a>
@@ Line 2,940: / Line 6,133: @@
 <p class = "date">07/2/13</p>
 <div align="center" style="border:1px solid white"><b>PCR of mlsR and Redo of C. sacc and C. beij PCR</b></div></br></br>
--------------------------pic-----------------------------------------
 <a href = "#top">Back to top</a>
@@ Line 2,972: / Line 6,164: @@
 <li>Contents of each tube re suspended in 500&#956;L of PCR dH<sub>2</sub>O</li>
 </br>
-----------------------------4PIC--------------------------------------------------
 </br>
 <a href = "#top">Back to top</a>
@@ Line 3,011: / Line 6,202: @@
 </table>
 </br>
----------------------------------------pic-----------------------------
 <table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
 <tr>
@@ Line 3,043: / Line 6,233: @@
 </table>
 </br>
-----------------------------PIc-------------------------------------------
 </br>
 <a href = "#top">Back to top</a>
@@ Line 3,092: / Line 6,282: @@
 <p>Procedure performed as outline on pg. 42</p>
 </br>
----------------------------3 pics------------------------------------------
 </br>
 <p>16S PCR of C. acetobutylicum, R. eutropha, C. sacchrolyticum, and C. beijerinki to confirm wheather or not we have what we think</p>
@@ Line 3,256: / Line 6,446: @@
 </table>
 </br>
--------------------------------------------pic---------------------------
 <p>We have bands of the correct length for Cb, Cs, and Ca, they are too faint for sequencing. So, we are going to redo the PCR without template dilutions and do 35 cycles instead of 30 cycles</p>
 </br>
@@ Line 3,615: / Line 6,805: @@
 <div align="center" style="border:1px solid white"><b>Gel of puc19 with KIVD Digest (EcoR1 and Pst1)</b></div></br></br>
 </br>
-----------------------------------pic-------------------------------------------------
 <a href = "#top">Back to top</a>
@@ Line 3,753: / Line 6,942: @@
 <div align="center" style="border:1px solid white"><b>Plasmid Isolation of pUC19 from BBa_k273006</b></div></br></br>
 </br>
-----------------------2pic-------------------------
 </br>
 <a href = "#top">Back to top</a>
@@ Line 3,849: / Line 7,037: @@
 </ol>
 </br>
--------------------------------pic-------------------------------------
 </br>
 <a href = "#top">Back to top</a>
@@ Line 3,860: / Line 7,047: @@
 <p class = "date">05/31/13</p>
 <div align="center" style="border:1px solid white"><b>Plasmid Extraction/Purification of Top 10 E. coli  Transformed with pSB1C3 and Clostridial Origin</b></div></br></br>
------------------------------11 PICS----------------------------------
 </br>
 <p>Digestion as preformed on pg. 47</p>
@@ Line 4,005: / Line 7,191: @@
 <div align="center" style="border:1px solid white"><b>Plasmid Isolation of pUC19 from BBa_k273006</b></div></br></br>
 </br>
-----------------------2pic-------------------------
 </br>
 <a href = "#top">Back to top</a>
@@ Line 4,101: / Line 7,286: @@
 </ol>
 </br>
--------------------------------pic-------------------------------------
 </br>
 <a href = "#top">Back to top</a>
@@ Line 4,112: / Line 7,296: @@
 <p class = "date">05/31/13</p>
 <div align="center" style="border:1px solid white"><b>Plasmid Extraction/Purification of Top 10 E. coli  Transformed with pSB1C3 and Clostridial Origin</b></div></br></br>
------------------------------11 PICS----------------------------------
 </br>
 <p>Digestion as preformed on pg. 47</p>
@@ Line 4,183: / Line 7,366: @@
 <p>Quantification with Nanodrop</p>
 </br>
-------------------------------------6 PICS-----------------------------
 </br>
 <table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
@@ Line 4,294: / Line 7,476: @@
 </table>
 </br>
-------------------------------------4PIC-------------------------------------
 </br>
 <table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;">
@@ Line 4,404: / Line 7,585: @@
 </table>
 </br>
-------------------------------------3 PIC-----------------------------------
 </br>
 <p>Used wrong kit (Spin mini prep). Suppose to use PCR purification kit. </p>\
@@ Line 4,489: / Line 7,669: @@
 </ul>
 </br>
------------------------------PIC-------------------------------------------------------------------------
 </br>
 <a href = "#top">Back to top</a>
@@ Line 4,577: / Line 7,756: @@
 <p>Nanodrop Quantification</p>
 </br>
----------------------------4 PICS---------------------------------------------------------
 <a href = "#top">Back to top</a>
@@ Line 4,792: / Line 7,970: @@
 <p>4&#956;L PCR dH<sub>2</sub>O</p>
 </br>
-------------------pic----------------------------------------------------------
 </br>
 <p>We didn’t get what we were trying to amplify</p>
@@ Line 4,823: / Line 8,000: @@
 <p>Repeat 30x</p>
 </br>
----------------------------------pic-----------------------------
 </br>
 <p>32ng/&#956;L of DNA template (pIKM1) successfully produced the Clostridial origin</p>
@@ Line 4,911: / Line 8,088: @@
 <li>well 4: negative control</li>
 </ul>
--------------------------------------pic---------------------------------------------
 </br>
 <p>The band we get from the gel is too small to be the origin that we want. We think that we have just been given something other than what we thought we had</p>
@@ Line 5,011: / Line 8,187: @@
 <p>2) Nanodrop quantification of plasmid DNA </p>
 </br>
------------------------------PIC-----------------------------------------------
 </br>
 <p>3) Digest of pIKM1 and pAN1 with RSA1
@@ Line 5,104: / Line 8,279: @@
 </div>
 </br>
--------------------------------------------------------------------PIC_________------------------
 </br>
 <a href = "#top">Back to top</a>
@@ Line 5,321: / Line 8,495: @@
 <li>Run gel for 45 minutes-1.5 hours at 85 volts
 </ol>
-------------------------PIC PG 15--------------------------------------------------------------------------
 <p>PCR Reaction Protocol</p>
 <ol>