Team:Heidelberg/Templates/DelH week15

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Revision as of 12:40, 25 October 2013

05-08 - 13-08-13

Generation of DelH Plasmid 01-08

Colony-PCR Conditions CP.W15.A

From colonies inoculated on 04-08

Reagent	3c	3c	3c	3c	3c	4a	4a	4a	4a	4a	6b	6b	6b	6b	6b
Expected length [kb]	663	663	663	663	663	663	663	663	663	663	663	663	663	663	663
Named	3c 1	3c 2	3c 3	3c 4	3c 5	4a 1	4a 2	4a 3	4a 4	4a 5	6b 1	6b 2	6b 3	6b 4	6b 5
Template (1 µl)	1 colonies of E3c	1 colonies of E3c	1 colonies of E3c	1 colonies of E3c	1 colonies of E3c	1 colonies of E4a	1 colonies of E4a	1 colonies of E4a	1 colonies of E4a	1 colonies of E4a	1 colonies of E6b	1 colonies of E6b	1 colonies of E6b	1 colonies of E6b	1 colonies of E6b
Primer fw 10 µM	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2
Primer rev 10 µM	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev
Dream-Taq Polymerase (2x)	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl
ddH₂O	5 µl	5 µl	5µl	5 µl	5 µl	5 µl	5 µl	5 µl	5 µl	5 µl	5 µl	5 µl	5µl	5 µl	5 µl	5 µl	5 µl	5 µl

Cycles	Temperature [°C]	Time [s]
1	95	120
12	95	60
	68 (touchdown -0.5°C)	30
	72	45
18	95	60
	65 (touchdown -0.5°C)	30
	72	45
1	12	inf

Result

Expected band: 663 bp

Fig.15.2 Gel of picked colonies with insert DelH-BB (loaded 20 µL of PCR)
l1-5: 5 seperate colonies which were together in 3a PCR earlier, l6: 2log
no bands = no colony positive

Fig.15.1 Gel of picked colonies with insert DelH-BB (loaded 20 µL of PCR)
l1-5: 5 seperate colonies which were together in 3a PCR earlier, l6: 2log, l7-11: 5 seperate colonies which were together in 3a PCR earlier,
no bands = no colony positive

None of the gels shows expected band.

=> None of the colonies is positive.

Miniprep

Of colonies 6b(1)-6b(5), 6a(1), 6a(2), 3c(3), a miniprep was performed.

Sample	Concentration [ng/µl]	µl avaiblable
6b(1)	33.8	18
6b(2)	33.4	18
6b(3)	36.9	18
6b(4)	59.6	18
6b(5)	18	18
6a(1)	60	18
6a(2)	62.3	18
3c(3)	40.9	18

Test Restriction Digest

Sample	Miniprep [µl]	CutSmart Buffer [µl]	NotI-HF [µl]	H₂O [µl]	Total [µl]
6b(1)	15	2	1	3	20
6b(2)	15	2	1	3	20
6b(3)	15	2	1	3	20
6b(4)	10	2	1	8	20
6b(5)	15	2	1	3	20
6a(1)	10	2	1	8	20
6a(2)	10	2	1	8	20
3c(3)	15	2	1	3	20

Result

Expected bands: 11,079 bp, 6,201 bp, 3,998 bp, 2,371 bp

Fig.15.3 test digest of listed colonies (loaded 20 µL of PCR)
l1-5: 5 seperate colonies of 6b, l6: 2log, l7 6a(1), l8 6a(2), l9 3c(3)
results are shown in table below

Expected bands	Present bands
11.079 bp	?
6.201 bp	no
3.998 bp	yes
2.371 bp	no

in silico PCR of BB shows the following result:

2 fragments generated:

1: 3.998 bp - From NotI[9] To NotI[4007]

2: 1.093 bp - From NotI[4007] To NotI[9]

But an additional band between 1-2 Kb is present!

=> Talk to Advisors, because it isn't what we expected. What could it be?

Possible next steps:

a) Wait for the new strain of D. acidovorans SHP1

b) Perform a re-PCR of minipreps (one with BB and one with DelH G0 fragment)

Generation of Backbone pSB6A1-lacI-mRFP

Restriction Digest with DpnI

After consulting the advisors, we decided not to continue the strategy with the old construct and wait with DelH until the new strain of D. acidovorans arrives. Until then, we digest the backbone (which is used also in following experiments) with DpnI to get rid of any remaining template.

Reagent	Amount [µl]
Gel extracted lineralized by PCR (template=10.4 Kb)	17
DpnI	1
CutSmart buffer	2
Total	20

1 h incubation at 37°C
20 min inactivation of enzyme by heat shock at 80°C for 20 min

Purification

As template, the PCR of 10,4 PCR, which was digested with DpnI on 06-08, was used.
Purification was performed with nucleotide removal kit (wrong column was used, but there was some yield at the nanodrop)

=> Again, checking on gel (2 µl + 8 µl 1xloading dye)

Fig.15.4 checking concentration of purified BB (PSB6A1-lacI-mRFP) (loaded 2 µL of PCR)
l1 2log ladder l2: pSB6A1-lacI-mRFP
l2 show expected band at 5 Kb

=> Concentration at nanodrop was 27.2 ng/µl

Amplification of DelH G0

PCR Conditions G0.W15.A

Reagent	Amount [µl]
Expected length [Kb]	18.521
Named	G0
Template	1 µl New strain culture SHP1 (resuspended pellet)
Primer fw 10 µM	1 µl short2
Primer rev 10 µM	1 µl HM08
Phusion Flash Ready Mix	10
ddH₂O	5
DMSO	1

Cycles	Temperature [°C]	Time [s]
1	98	5
30	98	1
	65	5
	72	4:45 min
1	72	10 min
1	12	inf

Lid preheated at 98°C
No hot start

Result

Expected band: 18.521 Kb

Fig.15.5 amplified fragment DelH-complete (loaded 20 µL of PCR)
l1: G0, l21Kb+ ladder
no band at 18 Kb only a specific band at 5 Kb, but that is not useful

Gel does not show the expected band.

=> Further improve PCR conditions.

PCR Conditions G0.W15.B

Reagent	Amount [µl]
Expected length [Kb]	18.521
Named	G0
Template	Picked colony
Primer fw 10 µM	1 µl short2
Primer rev 10 µM	1 µl HM08
Phusion Flash Ready Mix	10
ddH₂O	5
DMSO	1

Cycles	Temperature [°C]	Time [s]
1	98	5
30	98	1
	65	5
	72	4:45 min
1	72	10 min
1	12	inf

Lid preheated at 98°C
No hot start

=> Becaus it worked well we amplified 3x G0 with the same conditions.

PCR Conditions G0.W15.C

3x 20 µl PCR reactions were prepared

Reagent	Amount [µl]
Expected length [Kb]	18.521
Named	G0
Template	Picked colony
Primer fw 10 µM	1 µl short2
Primer rev 10 µM	1 µl HM08
Phusion Flash Ready Mix	10
ddH₂O	5
DMSO	1

Cycles	Temperature [°C]	Time [s]
1	98	5
30	98	1
	65	5
	72	4:45 min
1	72	10 min
1	12	inf

Lid preheated at 98°C
No hot start

Result

Expected band: 18 Kb

Fig.15.7 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1-3:3x G0 (complete fragment amplified with DN11 & HM08), l4: 1kb+ ladder, l5-6:2x G1/2a (complete fragment amplified with DN11 & HM06)
l1-3: band at 18 Kb was cut out l5-6: band at 13 Kb was cut out

Fig.15.6 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1-3:3x G0 (complete fragment amplified with DN11 & HM08), l4: 1kb+ ladder, l5-6:2x G1/2a (complete fragment amplified with DN11 & HM06)
l1-3: show the expected band at 18 Kb and l5-6: show the specific band at 13 Kb

Gel shows the expected bands.

=> Fragments were cut and gel extracted.

Amplification of DelH G1/2a

PCR Conditions G1/2a.W15.A

Reagent	Amount [µl]	Amount [µl]
Expected length [Kb]	13.083	13.083
Named	G1/2a	G1/2a
Template	Picked colony	Picked colony
Primer fw 10 µM	1 µl short2	1 µl short2
Primer rev 10 µM	1 µl HM06	1 µl HM06
Phusion Flash Ready Mix	10	10
ddH₂O	5	4
DMSO	1	0

Cycles	Temperature [°C]	Time [s]
1	98	5
30	98	1
	65	5
	72	4:45 min
1	72	10 min
1	12	inf

Lid preheated at 98°C
No hot start

Result

Fig.15.7 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1-3:3x G0 (complete fragment amplified with DN11 & HM08), l4: 1kb+ ladder, l5-6:2x G1/2a (complete fragment amplified with DN11 & HM06)
l1-3: band at 18 Kb was cut out l5-6: band at 13 Kb was cut out

Fig.15.6 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1-3:3x G0 (complete fragment amplified with DN11 & HM08), l4: 1kb+ ladder, l5-6:2x G1/2a (complete fragment amplified with DN11 & HM06)
l1-3: show the expected band at 18 Kb and l5-6: show the specific band at 13 Kb

Gel shows the expected bands.

=> Fragments were cut and gel extracted.

PCR Conditions G1/2a.W15.B

Reagent	Amount [µl]
Expected length [Kb]	13.083
Named	G1/2a
Template	Picked colony
Primer fw 10 µM	1 µl short2
Primer rev 10 µM	1 µl HM06
Phusion Flash Ready Mix	10
ddH₂O	5
DMSO	1

Cycles	Temperature [°C]	Time [s]
1	98	5
30	98	1
	65	5
	72	4:45 min
1	72	10 min
1	12	inf

Lid preheated at 98°C
No hot start

Result

Expected band: 13 Kb

Fig.15.9 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1-3:3x G0 (complete fragment amplified with DN11 & HM08), l4: 1kb+ ladder, l5-6:2x G1/2a (complete fragment amplified with DN11 & HM06)
l1-3: band at 18 Kb was cut out l5-6: band at 13 Kb was cut out

Fig.15.8 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1-3:3x G0 (complete fragment amplified with DN11 & HM08), l4: 1kb+ ladder, l5-6:2x G1/2a (complete fragment amplified with DN11 & HM06)
l1-3: show the expected band at 18 Kb and l5-6: show the specific band at 13 Kb

Gel shows the expected bands.

=> Fragments were cut and gel extracted.

Amplification of DelH G2b

PCR Conditions G2b.W15.A

Reagent	Amount [µl]	Amount [µl]
Expected length [Kb]	5.472	5.472
Named	G2b	G2b
Template	Picked colony	Picked colony
Primer fw 10 µM	1 µl HM07	1 µl HM07
Primer rev 10 µM	1 µl HM08	1 µl HM08
Phusion Flash Ready Mix	10	10
ddH₂O	5	4
DMSO	1	0

Cycles	Temperature [°C]	Time [s]
1	98	5
30	98	1
	68	5
	72	120
1	72	10 min
1	12	inf

Lid preheated at 98°C
No hot start

Result

Fig. 15.10: Gel of amplified DelH G2b fragment l1:2log, l2-3:G2b amplified

Summary of Gel Extracted Fragments

Concentrations (by nanodrop) are summarized in the following table and gel picture.

Fragment	Concentration [ng/µl]
G0	3.5
G1/2a	4.9
G2b	18.6

Fig.15.11 Analyse-gel of DelH-fragments (loaded 5 µL of G0 and G1/2a and 1 µl of G2b)
lane 1 1kb+ ladder, lane 2 G0 (complete DelH with DN11 & HM08), lane 3 G1/2a (part of DelH with DN11 & HM06), lane 4 G2b (part of DelH with HM07 & HM08)

Generation of DelH Plasmid 09-08

Gibson Assembly

We decided two assemble 2 different constructs and one control (only the backbone).

Construct	G0 [µl]	G1/2a [µl]	G2b [µl]	BB [µl]	2x Gibson Master Mix [µl]	Final amount [µl]
1	9	0	0	1	10	20
2	0	8	1	1	10	20
Control (C)	0	0	0	1 (+9 ddH₂O )	10	20

Incubated 1 h at 50°C

Electroporation

Of each Gibson assembly, 5 µl are diluted in 10 µl ddH₂O
The rest of the Gibson assembly is stored at -20°C

Conoly-PCR Conditions CP.W15.A

Picked 8 colonies from plates with 10µl and 100 µl construct 1, and plate with 100 µl of construct 2. 1 additional colony was picked from plate with 10 µl of construct 2 (because there were no more cells) and 1 colony of the control(100µl), and one colony of the control (100µl) with new electrocompetent cells.

In the following table construct 1 is named C1, and construct 2 = C2 and Control = Co

Reagent	C1 (10 µl)	C1 (10 µl)	C1 (10 µl)	C1 (10 µl)	C1 (10 µl)	C1 (10 µl)	C1 (10 µl)	C1 (10 µl)	C1 (100 µl)	C1 (100 µl)	C1 (100 µl)	C1 (100 µl)	C1 (100 µl)	C1 (100 µl)	C1 (100 µl)	C1 (100 µl)	C2 (10 µl)	C2 (10 µl)	C2 (10 µl)	C2 (10 µl)	C2 (10 µl)	C2 (10 µl)	C2 (10 µl)	C2 (10 µl)	C2 (100 µl)	Co (100 µl)	Co(100 µl)
Expected length [bp]	663	663	663	663	663	663	663	663	663	663	663	663	663	663	663	663	663	663	663	663	663	663	663	663	663	no product	no product
Named	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25	26	27
Primer fw 10 µM	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2
Primer rev 10 µM	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev
Dream-Taq Polymerase (2x)	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl
ddH₂O	6 µl	6 µl	6µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl

Cycles	Temperature [°C]	Time [s]
1	95	120
12	95	60
	68 (touchdown -0.5°C)	30
	72	45
18	95	60
	65 (touchdown -0.5°C)	30
	72	45
1	12	inf

Result

Expected band: 663 bp

Fig.15.12 screening PCR with DN07 & VF2 (loaded 1 µL of PCR)
l1:2log ladder,l2-17: colonies C1, l18-24: colonies C2, l25:2log ladder
None of the colonies showed expected band, instead, all have band at ~6 kb

Gel shows no banda at 663 bp. Instead, they show an unexpected band at ~6 Kb.

=> None of the colonies is positive.

Conoly-PCR Conditions CP.W15.B

Picked 20 colonies from plates with 10 µl and 100 µl construct 1 and construct 2 (80 colonies). 4 colonies were picked from the control (10 µl) and control (100 µl).

In the following table construct 1 is named C1, and construct 2 = C2 and Control = Co

Reagent	C1 (10 µl)	C1 (10 µl)	C1 (10 µl)	C1 (10 µl)	C1 (100 µl)	C1 (100 µl)	C1 (100 µl)	C1 (100 µl)
Expected length [bp]	663	663	663	663	663	663	663	663
Named	A	B	C	D	E	F	G	H
Primer fw 10 µM	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2
Primer rev 10 µM	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev
Dream-Taq Polymerase (2x)	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl
ddH₂O	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl

Reagent	C2 (10µl)	C2 (10µl)	C2 (10µl)	C2 (10µl)	C2 (100µl)	C2 (100µl)	C2 (100µl)	C2 (100µl)
Expected length [bp]	663	663	663	663	663	663	663	663
Named	I	J	K	L	M	N	O	P
Primer fw 10 µM	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2
Primer rev 10 µM	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev
Dream-Taq Polymerase (2x)	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl
ddH₂O	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl

Reagent	Co (10µl)	Co (10µl)	Co (10µl)	Co (10µl)	Co(100µl)	Co(100µl)	Co(100µl)	Co(100µl)
Expected length [bp]	663	663	663	663	663	663	663	663
Named	C1	C2	C3	C4	C5	C6	C7	C8
Primer fw 10 µM	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2	2 µl VF2
Primer rev 10 µM	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev	2 µl Screen_delH_rev
Dream-Taq Polymerase (2x)	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl	10 µl
ddH₂O	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl	6 µl

Cycles	Temperature [°C]	Time [s]
1	95	120
12	95	60
	68 (touchdown -0.5°C)	30
	72	45
18	95	60
	65 (touchdown -0.5°C)	30
	72	45
1	12	inf

Result

Expected band: 663 bp

Fig.15.13 screening PCR with DN07 & VF2 (loaded 1 µL of PCR)
l1-9: colonies A-I, l10:2log ladder, l11-17: colonies J-P, l12:2log ladder, l13-18: PCR performed with 6 colonies of the control plate
Neither the colonies A-P show the expected band nor the control shows any band

=> New Gibson Assembly will be performed

Gel shows no banda at 663 bp.

=> Neither the colonies A-P show the expected band, nor the control shows any band.

Generation of DelH Plasmid 11-08

Gibson Assembly

Because of the past results, we assume that the colonies can grow because the backbone religates. We also found out that the first overhang of the backbone has some homologity with a sequence at the end and we can assume that with Gibson, it primes itself.
So in the following step we will use less backbone. We prepared an 1:10 dilution of the backbone construct.

Reagent	Concentration [ng/µl]	Amount [µl] Mix A	Amount [µl] Mix B	Amount [µl] Mix C
G0	3.5	9	0	0
BB	1.8	1	1	1
Gibson Assembly Master Mix	2x	10	10	0

The time constant for the electroporation was unexpectedly low, hopefully high enough...

@@ Line 1: / Line 1: @@
 ==05-08 - 13-08-13 ==
 ===Generation of DelH Plasmid 01-08===
@@ Line 45: / Line 46: @@
 Expected band: 663 bp
 <br/>
-[[File:Heidelberg_20130805 colonyPCR3c-4a.png|200px|thumb|right|'''Fig.15.1''' Gel of picked colonies with insert DelH-BB (loaded 20 µL of PCR) <br> ''l1-5:'' 5 seperate colonies which were together in 3a PCR earlier, ''l6:'' 2log, ''l7-11:'' 5 seperate colonies which were together in 3a PCR earlier,  <br>  no bands = no colony positive]]
 [[File:Heidelberg_20130805 colonyPCR6b.png|200px|thumb|right|'''Fig.15.2''' Gel of picked colonies with insert DelH-BB (loaded 20 µL of PCR) <br> ''l1-5:'' 5 seperate colonies which were together in 3a PCR earlier, ''l6:'' 2log  <br>  no bands = no colony positive]]
-<div style="clear:both"></div>
+<div class="tright" style="clear:none">[[File:Heidelberg_20130805 colonyPCR3c-4a.png|200px|thumb|right|'''Fig.15.1''' Gel of picked colonies with insert DelH-BB (loaded 20 µL of PCR) <br> ''l1-5:'' 5 seperate colonies which were together in 3a PCR earlier, ''l6:'' 2log, ''l7-11:'' 5 seperate colonies which were together in 3a PCR earlier,  <br>  no bands = no colony positive]]</div>
 None of the gels shows expected band.
 :=> None of the colonies is positive.
 <br/>
+<div style="clear:both"></div>
 ====Miniprep====
 Of colonies 6b(1)-6b(5), 6a(1), 6a(2), 3c(3), a miniprep was performed.
@@ Line 98: / Line 101: @@
 <br/>
 [[File:Heidelberg_20130805 testdigest.png|200px|thumb|right|'''Fig.15.3''' test digest of listed colonies (loaded 20 µL of PCR) <br> ''l1-5:'' 5 seperate colonies of 6b, ''l6:'' 2log, ''l7'' 6a(1), ''l8'' 6a(2), ''l9'' 3c(3)  <br> results are shown in table below]]
-<div style="clear:both"></div>
 {| class="wikitable"
@@ Line 120: / Line 122: @@
 Possible next steps:
 :a) Wait for the new strain of ''D. acidovorans'' SHP1
 :b) Perform a re-PCR of minipreps (one with BB and one with DelH G0 fragment)
 <br/>
+<div style="clear:both"></div>
 ===Generation of Backbone pSB6A1-lacI-mRFP===
 ====Restriction Digest with DpnI====
@@ Line 147: / Line 150: @@
 => Again, checking on gel (2 µl + 8 µl 1xloading dye) <br/>
 [[File:Heidelberg_20130808 pSB6A1 2log.png|200px|thumb|right|'''Fig.15.4''' checking concentration of purified BB (PSB6A1-lacI-mRFP) (loaded 2 µL of PCR) <br>''l1'' 2log ladder ''l2:'' pSB6A1-lacI-mRFP  <br>  ''l2'' show expected band at 5 Kb ]]
-<div style="clear:both"></div>
 :=> Concentration at nanodrop was 27.2 ng/µl
 <br/>
+<div style="clear:both"></div>
 ===Amplification of DelH G0===
 ====PCR Conditions G0.W15.A====
@@ Line 195: / Line 199: @@
 <br/>
 [[File:Heidelberg_20130807 G0.png|200px|thumb|right|'''Fig.15.5''' amplified fragment DelH-complete (loaded 20 µL of PCR) <br>''l1:'' G0, ''l2''1Kb+ ladder <br>  no band at 18 Kb only a specific band at 5 Kb, but that is not useful]]
-<div style="clear:both"></div>
 Gel does not show the expected band.
 :=> Further improve PCR conditions.
 <br/>
+<div style="clear:both"></div>
 ====PCR Conditions G0.W15.B====
 {| class="wikitable" style="float:left; margin-right:1em"
@@ Line 236: / Line 241: @@
 | 1  || 12 || inf
 |}
-<div style="clear:both"></div>
 * Lid preheated at 98°C
 * No hot start
 :=> Becaus it worked well we amplified 3x G0 with the same conditions.
+<div style="clear:both"></div>
 ====PCR Conditions G0.W15.C====
 x 20 µl PCR reactions were prepared
@@ Line 278: / Line 284: @@
 | 1  || 12 || inf
 |}
-<div style="clear:both"></div>
 * Lid preheated at 98°C
 * No hot start
+<div style="clear:both"></div>
 ====Result====
 Expected band: 18 Kb
 <br/>
-[[File:Heidelberg_20130808 3xG0 2xG1-2a.png|200px|thumb|right|'''Fig.15.6''' gel of amplified DelH-fragment (loaded 20 µL of PCR) <br> ''l1-3:''3x G0 (complete fragment amplified with DN11 & HM08), ''l4:'' 1kb+ ladder, ''l5-6:''2x G1/2a (complete fragment amplified with DN11 & HM06) <br> ''l1-3:'' show the expected band at 18 Kb and ''l5-6:'' show the specific band at 13 Kb]]
+[[File:Heidelberg_20130808 3xG0 2xG1-2a cut.png|200px|thumb|right|'''Fig.15.7''' gel of amplified DelH-fragment (loaded 20 µL of PCR) <br> ''l1-3:''3x G0 (complete fragment amplified with DN11 & HM08), ''l4:'' 1kb+ ladder, ''l5-6:''2x G1/2a (complete fragment amplified with DN11 & HM06) <br> ''l1-3:'' band at 18 Kb was cut out ''l5-6:''  band at 13 Kb was cut out]]
- [[File:Heidelberg_20130808 3xG0 2xG1-2a cut.png|200px|thumb|right|'''Fig.15.7''' gel of amplified DelH-fragment (loaded 20 µL of PCR) <br> ''l1-3:''3x G0 (complete fragment amplified with DN11 & HM08), ''l4:'' 1kb+ ladder, ''l5-6:''2x G1/2a (complete fragment amplified with DN11 & HM06) <br> ''l1-3:'' band at 18 Kb was cut out ''l5-6:''  band at 13 Kb was cut out]]
+<div class="tright" style="clear:none">[[File:Heidelberg_20130808 3xG0 2xG1-2a.png|200px|thumb|right|'''Fig.15.6''' gel of amplified DelH-fragment (loaded 20 µL of PCR) <br> ''l1-3:''3x G0 (complete fragment amplified with DN11 & HM08), ''l4:'' 1kb+ ladder, ''l5-6:''2x G1/2a (complete fragment amplified with DN11 & HM06) <br> ''l1-3:'' show the expected band at 18 Kb and ''l5-6:'' show the specific band at 13 Kb]]</div>
-<div style="clear:both"></div>
 Gel shows the expected bands.
 :=> Fragments were cut and gel extracted.
 <br/>
+<div style="clear:both"></div>
 ===Amplification of DelH G1/2a===
 ====PCR Conditions G1/2a.W15.A====
@@ Line 328: / Line 336: @@
 | 1  || 12 || inf
 |}
-<div style="clear:both"></div>
 * Lid preheated at 98°C
 * No hot start
-====Result====
-[[File:Heidelberg_20130808 3xG0 2xG1-2a.png|200px|thumb|right|'''Fig.15.6''' gel of amplified DelH-fragment (loaded 20 µL of PCR) <br> ''l1-3:''3x G0 (complete fragment amplified with DN11 & HM08), ''l4:'' 1kb+ ladder, ''l5-6:''2x G1/2a (complete fragment amplified with DN11 & HM06) <br> ''l1-3:'' show the expected band at 18 Kb and ''l5-6:'' show the specific band at 13 Kb]]
- [[File:Heidelberg_20130808 3xG0 2xG1-2a cut.png|200px|thumb|right|'''Fig.15.7''' gel of amplified DelH-fragment (loaded 20 µL of PCR) <br> ''l1-3:''3x G0 (complete fragment amplified with DN11 & HM08), ''l4:'' 1kb+ ladder, ''l5-6:''2x G1/2a (complete fragment amplified with DN11 & HM06) <br> ''l1-3:'' band at 18 Kb was cut out ''l5-6:''  band at 13 Kb was cut out]]
 <div style="clear:both"></div>
+====Result====
+[[File:Heidelberg_20130808 3xG0 2xG1-2a cut.png|200px|thumb|right|'''Fig.15.7''' gel of amplified DelH-fragment (loaded 20 µL of PCR) <br> ''l1-3:''3x G0 (complete fragment amplified with DN11 & HM08), ''l4:'' 1kb+ ladder, ''l5-6:''2x G1/2a (complete fragment amplified with DN11 & HM06) <br> ''l1-3:'' band at 18 Kb was cut out ''l5-6:''  band at 13 Kb was cut out]]
+<div class="tright" style="clear:none">[[File:Heidelberg_20130808 3xG0 2xG1-2a.png|200px|thumb|right|'''Fig.15.6''' gel of amplified DelH-fragment (loaded 20 µL of PCR) <br> ''l1-3:''3x G0 (complete fragment amplified with DN11 & HM08), ''l4:'' 1kb+ ladder, ''l5-6:''2x G1/2a (complete fragment amplified with DN11 & HM06) <br> ''l1-3:'' show the expected band at 18 Kb and ''l5-6:'' show the specific band at 13 Kb]]</div>
 Gel shows the expected bands.
 :=> Fragments were cut and gel extracted.
 <br/>
+<div style="clear:both"></div>
 ====PCR Conditions G1/2a.W15.B====
 {| class="wikitable" style="float:left; margin-right:1em"
@@ Line 375: / Line 386: @@
 | 1  || 12 || inf
 |}
-<div style="clear:both"></div>
 * Lid preheated at 98°C
 * No hot start
+<div style="clear:both"></div>
 ====Result====
 Expected band: 13 Kb
 <br/>
-[[File:Heidelberg_20130808 3xG0 2xG1-2a.png|200px|thumb|right|'''Fig.15.8''' gel of amplified DelH-fragment (loaded 20 µL of PCR) <br> ''l1-3:''3x G0 (complete fragment amplified with DN11 & HM08), ''l4:'' 1kb+ ladder, ''l5-6:''2x G1/2a (complete fragment amplified with DN11 & HM06) <br> ''l1-3:'' show the expected band at 18 Kb and ''l5-6:'' show the specific band at 13 Kb]]
+[[File:Heidelberg_20130808 3xG0 2xG1-2a cut.png|200px|thumb|right|'''Fig.15.9''' gel of amplified DelH-fragment (loaded 20 µL of PCR) <br> ''l1-3:''3x G0 (complete fragment amplified with DN11 & HM08), ''l4:'' 1kb+ ladder, ''l5-6:''2x G1/2a (complete fragment amplified with DN11 & HM06) <br> ''l1-3:'' band at 18 Kb was cut out ''l5-6:''  band at 13 Kb was cut out]]
- [[File:Heidelberg_20130808 3xG0 2xG1-2a cut.png|200px|thumb|right|'''Fig.15.9''' gel of amplified DelH-fragment (loaded 20 µL of PCR) <br> ''l1-3:''3x G0 (complete fragment amplified with DN11 & HM08), ''l4:'' 1kb+ ladder, ''l5-6:''2x G1/2a (complete fragment amplified with DN11 & HM06) <br> ''l1-3:'' band at 18 Kb was cut out ''l5-6:''  band at 13 Kb was cut out]]
+<div class="tright" style="clear:none">[[File:Heidelberg_20130808 3xG0 2xG1-2a.png|200px|thumb|right|'''Fig.15.8''' gel of amplified DelH-fragment (loaded 20 µL of PCR) <br> ''l1-3:''3x G0 (complete fragment amplified with DN11 & HM08), ''l4:'' 1kb+ ladder, ''l5-6:''2x G1/2a (complete fragment amplified with DN11 & HM06) <br> ''l1-3:'' show the expected band at 18 Kb and ''l5-6:'' show the specific band at 13 Kb]]</div>
-<div style="clear:both"></div>
 Gel shows the expected bands.
 :=> Fragments were cut and gel extracted.
 <br/>
+<div style="clear:both"></div>
 ===Amplification of DelH G2b===
 ====PCR Conditions G2b.W15.A====
@@ Line 425: / Line 439: @@
 | 1  || 12 || inf
 |}
-<div style="clear:both"></div>
 * Lid preheated at 98°C
 * No hot start
+<div style="clear:both"></div>
 ====Result====
 [[File:Heidelberg_20130808 2log 2xDelHG2b.png|200px|thumb|'''Fig. 15.10''': Gel of amplified DelH G2b fragment ''l1:''2log, ''l2-3:''G2b amplified]]
@@ Line 511: / Line 526: @@
 <br/>
 [[File:Heidelberg_20130810_Colony_PCR_DelH_Gel1_2.png|200|thumb|right|'''Fig.15.12''' screening PCR with DN07 & VF2 (loaded 1 µL of PCR) <br> ''l1:''2log ladder,''l2-17:'' colonies C1, ''l18-24:'' colonies C2, ''l25:''2log ladder <br> None of the colonies showed expected band, instead, all have band at ~6 kb]]
-<div style="clear:both"></div>
 Gel shows no banda at 663 bp. Instead, they show an unexpected band at ~6 Kb.
 :=> None of the colonies is positive.
 <br/>
+<div style="clear:both"></div>
 ====Conoly-PCR Conditions CP.W15.B====
 Picked 20 colonies from plates with 10 µl and 100 µl construct 1 and construct 2 (80 colonies). 4 colonies were picked from the control (10 µl) and control (100 µl).
@@ Line 593: / Line 608: @@
 [[File:Heidelberg_20130811 SCREENINGpcrA-P.png|thumb|right|'''Fig.15.13''' screening PCR with DN07 & VF2 (loaded 1 µL of PCR) <br>''l1-9:'' colonies A-I, ''l10:''2log ladder, ''l11-17:'' colonies J-P, ''l12:''2log ladder, ''l13-18:'' PCR performed with 6 colonies of the control plate <br>  Neither the colonies A-P show the expected band nor the control shows any band]]
 :=> New Gibson Assembly will be performed
-<div style="clear:both"></div>
 Gel shows no banda at 663 bp.
 :=> Neither the colonies A-P show the expected band, nor the control shows any band.
 <br/>
+<div style="clear:both"></div>
 ===Generation of DelH Plasmid 11-08===
 ====Gibson Assembly====

Team:Heidelberg/Templates/DelH week15

From 2013.igem.org

Revision as of 12:40, 25 October 2013

Contents

05-08 - 13-08-13

Generation of DelH Plasmid 01-08

Colony-PCR Conditions CP.W15.A

Result

Miniprep

Test Restriction Digest

Result

Generation of Backbone pSB6A1-lacI-mRFP

Restriction Digest with DpnI

Purification

Amplification of DelH G0

PCR Conditions G0.W15.A

Result

PCR Conditions G0.W15.B

PCR Conditions G0.W15.C

Result

Amplification of DelH G1/2a

PCR Conditions G1/2a.W15.A

Result

PCR Conditions G1/2a.W15.B

Result

Amplification of DelH G2b

PCR Conditions G2b.W15.A

Result

Summary of Gel Extracted Fragments

Generation of DelH Plasmid 09-08

Gibson Assembly

Electroporation

Conoly-PCR Conditions CP.W15.A

Result

Conoly-PCR Conditions CP.W15.B

Result

Generation of DelH Plasmid 11-08

Gibson Assembly