Team:OU-Norman OK/Project/Notebook

From 2013.igem.org

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


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


HTML tutorial


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


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


  1. Transform 4μL of ligation product into 100μL aliquots of competent E. coli cells
  2. Incubate on ice for 30 minutes
  3. Heat shock in water bath for 60 seconds
  4. Incubate on ice for 5 minutes
  5. Add 600μL of psi broth
  6. Incubate at 37°C for 2 hours at 200 rpm
  7. 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


HTML tutorial


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08/21/13

Transformation of Ter + pSB1C3 and Adh6 + pSB1C3


  1. Thaw competent Top10 cell of E. coli from -80°C (100μL aliquots)
  2. Add 20μL of ligation product to Top 10 E. coli
  3. Incubate on ice for 30 minutes
  4. Heat shock in water bath for exactly 60 seconds
  5. Incubate on ice for 5 minutes
  6. Add 600μL of psi broth
  7. Incubate at 37°C for 2 hours at 200 rpm
  8. Make dilutions using 90μL of psi broth: 1:10, 1:100, 1:1000
  9. 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 Back to top

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


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


  1. Thaw competent 100μL aliquot of E. coli cells from -80°C freezer
  2. Add 20μL of ligation poduct to Top10 E. coli
  3. Incubate on ice for 30 minutes
  4. Heat shock in water bath for 60 seconds
  5. Incubate on ice for 5 minutes
  6. Add 600μL of psi broth
  7. Incubate at 37°C for 2 hours at 200 rpm
  8. 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&#956L85.9ng) = 6.0μL

#15 Aadc+ 500ng (1&#956L117.5ng) = 4.5μL

#20 Aadc+ 500ng (1&#956L92.1ng) = 5.5μL

#23 Aadc- 500ng (1&#956L184.8ng) = 3.0μL

#6 Aadc- 500ng (1&#956L218.1ng) = 2.5μL

#17 thl+ 500ng (1&#956L100.0ng) = 5.0μL

#3 thl+ 500ng (1&#956L84.4ng) = 6.0μL

#10 thl+ 500ng (1&#956L90.4ng) = 5.5μL

#14 thl- 500ng (1&#956L85.7ng) = 6.0μL

#21 thl- 500ng (1&#956L89.1ng) = 5.5μL

#4 thl- 500ng (1&#956L131.8ng) = 4.0μL

#9 Ptb+ 500ng (1&#956L81.4ng) = 6.0μL

#1 Ptb+ 500ng (1&#956L124.2ng) = 4.0μL

#26 Ptb+ 500ng (1&#956L206.7ng) = 2.5μL

#18 Ptb- 500ng (1&#956L103.7ng) = 5.0μL

#8 Ptb- 500ng (1&#956L150.1ng) = 3.5μL

#5 Ptb- 500ng (1&#956L216.0ng) = 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

HTML tutorial

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

HTML tutorial


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08/1/13

Restriction Digest using EcoR1 and Pst1 (26 July Quantification)


#16-6D 500ng (1&#956L108.6ng) = 4.5μL

#14-20E 500ng (1&#956L235.2ng) = 2.0μL

#12-20G 500ng (1&#956L135.1ng) = 3.5μL

#8-20G 500ng (1&#956L192.3ng) = 2.5μL

#14-20A 500ng (1&#956L156.5ng) = 3.0μL

#9-6D 500ng (1&#956L`99.7ng) = 2.5μL

#10-4P 500ng (1&#956L131.8ng) = 4.0μL

#7-20G 500ng (1&#956L232.8ng) = 2.0μL

#3-23B 500ng (1&#956L90.0ng) = 5.5μL

#16-23B 500ng (1&#956L123.5ng) = 4.0μL

#9-4P 500ng (1&#956L145.2ng) = 3.5μL

#9-20G 500ng (1&#956L106.8ng) = 4.5μL

#15-20A 500ng (1&#956L150.1ng) = 3.5μL

#11-20E 500ng (1&#956L126.2ng) = 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

HTML tutorial


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08/01/13

Ampicillin 1x Preparation


To make 40mL of ampicillin 1x antibiotic

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

    1. 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


      HTML tutorial


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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)


      HTML tutorial

      HTML tutorial


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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&#956L58.1ng) = 8.5μL

      #9-4P 500ng (1&#956L40.2ng) = 12.5μL

      #14-20A 500ng (1&#956L43.5ng) = 12.5μL

      #7-20G 500ng (1&#956L59.4ng) = 12.5μL

      #14-20E 500ng (1&#956L37.5ng) = 12.5μL

      #10-4P 500ng (1&#956L43.6ng) = 12.5μL

      #15-20A 500ng (1&#956L51.6ng) = 12.5μL

      #16-23B 500ng (1&#956L52.5ng) = 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

      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

      HTML tutorial


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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.


      HTML tutorial


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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


      HTML tutorial


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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

      HTML tutorial


      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

      HTML tutorial


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      07/24/13

      DNA Extraction of R. eutropha


      1. Re suspend cells in 500μL 1x STE buffer
      2. Vortex until mixture is homogenous
      3. Add 50μL of 10% Sodium-dodecyl sulfate (SDS)
      4. IF cells insufficiently lysed, 2 minutes in bead beater
      5. Add 500μL of Chloroform:Phenol pH 8
        • Extract bottom layer (2 phases present)
        • Minimize time the chloroform is out of fridge
      6. Vortex
      7. Centrifuge for 30 seconds
        • 14000 rpm
      8. Transfer top phase to 1.5mL microcentrifuge tube
        • Don't take up white precipitant from interphase of solution
      9. Add 500μL of isoamyl acetate (CIAA 24:1) to top phase
      10. Vortex
      11. Centrifuge for 30 seconds
        • 14000 rpm
      12. Transfer top phase to 1.5mL microcentrifuge tube
        • Don't take up white precipitant from interphase of solution
      13. REPEAT STEPS 9-12
        • Note: Keep track of amount finally transfered in step 12
      14. Add (2/10) volume (amount of top phase) of 3M Sodium Acetate
      15. Add (7/10) volume (step 14 amount) of Isopropanol
        • Note: Mix solutions with pipette
      16. Centrifuge for 25 minutes
        • 14000 rpm
      17. Dispose of liquid portion
        • Don't disturb invisible pellet
      18. Dry pellet on heat block at 60°C for 10 minutes
        • Leave caps open for evaporation of isopropanol
      19. Re suspend pellet in 50μL of distilled water

      HTML tutorial


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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


      HTML tutorial

      HTML tutorial

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      07/23/13

      Digest of Cc Adh and Cs Adh


      500ng DNA for each reaction


      500ng (1&#956L39.7ng) = 12.5 μL Cc DNA

      500ng (1&#956L36.0ng) = 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&#956L202ng) = 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


      HTML tutorial

      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

      HTML tutorial

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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

      HTML tutorial

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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)

      HTML tutorial

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      07/22/13

      Plasmid Extraction of Ca pSOL


      Used Qia Quick Prep Plasmid Extraction Kit


      HTML tutorial

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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

      HTML tutorial

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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&#956L112.2ng) = 4.45 μL → 4.5 μL

      500ng (1&#956L124.5ng) = 4.02 μL → 4.0 μL

      500ng (1&#956L117.7ng) = 4.25 μL → 4.5 μL

      500ng (1&#956L162.0ng) = 3.09 μL → 3.0 μL

      500ng (1&#956L188.7ng) = 2.65 μL → 2.5 μL

      500ng (1&#956L206.4ng) = 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


      HTML tutorial



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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


      HTML tutorial



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      07/19/13

      PCR of Ccel Adh


      Preformed PCR to amplify Adh out of C. cellulolyticum


      HTML tutorial



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      07/19/13

      PCR of Cb SAdh


      Choose lower temperature (50°C) for annealing.


      HTML tutorial



      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.


      HTML tutorial



      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 Total
      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


      HTML tutorial


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      07/17/13

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


      HTML tutorial


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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]


      1. For 5mL tubes, each containing 3mL of meat extract broth inoculated with R. eutropha cells, which were in stationary phase
      2. 1mL aliquots transferred stepwise to two 15mL epi tubes, spinning at 14,000 rpm for five minutes to pellet cells
      3. Supernatant discarded and additional 1mL aliquot added, spun until all cells pelleted
      4. Cells re suspended in 500μL if 1x STE buffer in each of the two epi tubes
      5. Vortex
      6. 50μL of 10% SDS added to each tube
      7. 500μL of TE saturated phenol pH8 added to each tube
      8. Vortex well and centrifuge for 30 seconds
      9. Aqueous recovered
      10. Phenol extraction
      11. Steps 7-9 repeated
      12. 500μL chloroform added to each tube and vortexed
      13. Aqueous discarded
      14. Chloroform extraction repeated
      15. 400μL split into each of four tubes
      16. 100μL of 3M NaOH added to each tube
      17. 350μL isopropanol added to each tube
      18. Centrifuge at 14000 rpm for 25 minutes
      19. Liquid discarded
      20. Tubes places on 80°C heating block for 15 minutes
      21. Contents of each tube re suspended in 500μL of PCR dH2O


      22. Back to top

        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
        50μL for each reaction, three reactions for each organism with different template dilutions

        • 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)


        0.15g
        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
        1. Cycle chamber airlock; twice
        2. Clean gloves and chamber with 1N HCl (x2)
        3. ???????????????????
        4. Use sterile syringe to add 0.5mL media to inner vial
        5. Mix with syringe
        6. Add 0.5 of inoculums to each of the two media vials
        7. 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

        AdhE1 = 2577 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

        Back to top

        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

        1. Re suspend cells in 500μL 1x STE buffer
        2. Vortex
        3. Add 1/10 volume of 10% SDS
        4. 2 minutes in bead beater
        5. 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
        6. Vortex
        7. Centrifuge for 30 seconds
        8. Recover aqueous phase
          • Don’t recover white material at interphase
          • Throw away
        9. Add 500μL Chloroform: Isoamyl acetate
        10. Vortex
        11. Centrifuge for 30 seconds
        12. Recover and throw away aqueous phase
        13. Repeat Steps 8-12
        14. Add 2/10 volumes 3M sodium acetate
        15. Add 7/10 volumes isopropanol
        16. Mix well
        17. Centrifuge for 25 minutes
        18. Remove liquid and don’t disturb invisible pellet
        19. Dry pellet at 60°C in heat block for 10 minutes
          • Leave caps off to allow isopropanol to evaporate
        20. 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


        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

        AdhE1 = 2577 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

        1. Re suspend cells in 500μL 1x STE buffer
        2. Vortex
        3. Add 1/10 volume of 10% SDS
        4. 2 minutes in bead beater
        5. 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
        6. Vortex
        7. Centrifuge for 30 seconds
        8. Recover aqueous phase
          • Don’t recover white material at interphase
          • Throw away
        9. Add 500μL Chloroform: Isoamyl acetate
        10. Vortex
        11. Centrifuge for 30 seconds
        12. Recover and throw away aqueous phase
        13. Repeat Steps 8-12
        14. Add 2/10 volumes 3M sodium acetate
        15. Add 7/10 volumes isopropanol
        16. Mix well
        17. Centrifuge for 25 minutes
        18. Remove liquid and don’t disturb invisible pellet
        19. Dry pellet at 60°C in heat block for 10 minutes
          • Leave caps off to allow isopropanol to evaporate
        20. 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



        DNA
        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

        b>


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 H2O

pSB1A3

10ng x μL/40ng = 1 : 4μL Dilution 10μL : 30μL PCR H2O

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

  1. 95°C for 45 seconds
  2. 52°C for 45 seconds
  3. 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
    Back to top

    04/19/13

    PCR of Clostridial Origin with New Primers

    1. 95°C for 45 seconds
    2. 52°C for 45 seconds
    3. 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 dH2O

    pSB1A3

    100ng x μL/75ng& = 1.6μL => 1.5μL + 8.5μL dH2O

    pSB1C3

    100ng x μL/43ng& = 1.6μL => 2.5μL + 7.5μL dH2O

    High fidelity aliquots of 100μL for PCR


    PCR

    1. 95°C for 2 minutes
    2. 95°C for 30 seconds
    3. 55°C for 30 seconds
    4. 68°C for 3 minutes
    5. 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


    Back to top

    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

    1. Highlight stage of interest (in this case; annealing)
    2. Selection "options"
    3. 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

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

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    03/13/13

    Making and Preparing Agarose Gel

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


    Running Gel of Post-Digested pSB1C3 and pSB1A3

    1. Move gel into gel rig container, pour 1x TAE buffer until it covers the gel surface
    2. Remove comb slowly
    3. Mix 5-10μL of digest with 3μL of 1:4 EZ Vision dye (Note: if using undigested DNA, only use 2μL
    4. 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
    5. 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

    1. Combine the following

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

    Regular PCR Cycle

    1. 95°C for 45 seconds
    2. 55°C for 1 minute
    3. 75°C for 1.5 minutes
    4. 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


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    03/8/13

    Nanodrop DNA Quanitification

    Protocol

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

    pSB1K3

    p34KM

    pIKM1

    pSB1A3

    pSB1C3


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    02/13/13

    Colonies were counted

    1:1000 dilution of AmpicillinR 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

    1. TOP10 competent cells in 100μL alliquots (x5) were thawed on ice and resuspended.
    2. 100ng of plasmid were added to cells
    3. Cells were placed on ice for 20 minutes
    4. Cells were transformed to a 42°C waterbath for 60 seconds
    5. After 60 seconds in waterbath, add 600μL of Psi proth IMMEDIATELY to clls
    6. Cells were incubated for 60 minutes at 37°C while shaking at 200rpm
    7. Dilutions of transformation mixture were made at 1:10, 1:100, and 1:1000
    8. 50mL of each dilution was plated on an LB + Antibiotic plate
    9. 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 (dH2O)

    pSB1C3

    10ng/μL×μL/43ng×20μL = 4.65μL (original plasmid concentration) + 15.35μL (dH2O)

    pSB1A3

    10ng/μL×μL/75ng×20μL = 2.6μL (original plasmid concentration) + 17.4μL (dH2O)

    pSB1K3

    10ng/μL×μL/40ng×20μL = 5.0μL (original plasmid concentration) + 15.0μL (dH2O)

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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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