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Revision as of 13:45, 22 October 2013 (view source) FloSmi (Talk | contribs) ← Older edit		Latest revision as of 15:34, 22 October 2013 (view source) FloSmi (Talk | contribs)
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	| rowspan="3" | 12 || 95 || 30		| rowspan="3" | 12 || 95 || 30
	|-		|-
-	| 68 (touchdown -0.5°C) || 30	+	| 68 ↓ 0.5 || 30
	|-		|-
	| 72 || 45		| 72 || 45
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	| rowspan="3" | 12 || 98 || 1		| rowspan="3" | 12 || 98 || 1
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-	| 68 (touchdown -0,5°C) || 5	+	| 68 ↓ 0.5 || 5
	|-		|-
	| 72 || 1:25		| 72 || 1:25
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	| rowspan="3" | 12 || 98 || 1		| rowspan="3" | 12 || 98 || 1
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-	| 68 (touchdown -0,5°C) || 5	+	| 68 ↓ 0.5 || 5
	|-		|-
	| 72 || 1:25		| 72 || 1:25

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Latest revision as of 15:34, 22 October 2013

Contents 1 30-09 - 06-10-13 1.1 Generation of DelH Plasmid pHM04 15-09 1.1.1 Colony-PCR Conditions CP.W23.A 1.1.2 Result 1.1.3 Amplification of backbone pSB6A1 for strategy pFS02 1.1.4 Results 1.1.5 Amplification of backbone pSB6A1 for strategy pFS02 1.1.6 Result

30-09 - 06-10-13

Generation of DelH Plasmid pHM04 15-09

Colony-PCR Conditions CP.W23.A

Template	23x 1 picked colony, positive control
Expected length [bp]	663
Named	1A - 12H
Primer fw 10 µM	100x 2 µl VF2
Primer rev 10 µM	100x 2 µl DN07
iTaq Polymerase (2x)	100x 10 µl
ddH2O	96x 6 µl

Cycles	Temperature DelH [°C]	Time [s]
1	95	120
12	95	30
	68 ↓ 0.5	30
	72	45
18	95	30
	65	30
	72	45
1	12	inf

Result

Expected band: 663 bp
[[|200px|thumb|right|Fig.23.1 Colony PCR of Gibson assembled DelH-BB without mRFP 23 colonies]]

Amplification of backbone pSB6A1 for strategy pFS02

Reagent	Amount
Template	1 µl of minipreped pSB6A1 (2L)
Primer fw 10 µM	2 µl FS78
Primer rev 10 µM	2 µl HM17
Phusion Flash Ready Mix	10 µl
ddH2O	5 µl

Cycles	Temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	1:25
18	98	1
	68	5
	72	1:25
1	72	5 min
1	4	inf

Cycles	Temperature [°C]	Time [s]
1	98	5
12	98	1
	58	5
	72	1:25
1	72	5 min
1	4	inf

Results

Amplification of backbone pSB6A1 for strategy pFS02

Reagent	Amount
Template	1 µl of minipreped pSB6A1 (2L)
Primer fw 10 µM	2 µl FS85
Primer rev 10 µM	2 µl HM17
Phusion Flash Ready Mix	10 µl
ddH2O	5 µl

Cycles	Temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	1:25
18	98	1
	68	5
	72	1:25
1	72	5 min
1	4	inf

Cycles	Temperature [°C]	Time [s]
1	98	5
12	98	1
	58	5
	72	1:25
1	72	5 min
1	4	inf

Result