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From 2013.igem.org

Contents 1 Tyrocidine-Indigoidine-Fusion - Standardization of Constructs 1.1 Amplification 1.2 Results 1.3 CPEC

Tyrocidine-Indigoidine-Fusion - Standardization of Constructs

So far, the Tyrocidine-Indigoidine-fusion constructs had two illegal cutting sites of RFC-10 restriction enzymes. Hence, we tried a CPEC-approach for which we reamplified a fragment from the vector with primers that introduced a mutation at the desired position.

Amplification

what	µl
pPW05 (dil.)	1
RB68	2
RB69	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	59	0:10
	72	3:00
1	72	10:00
1	10	inf

Results

CPEC

After gel-extraction of the large fragment, the following procedure was followed:

• 22µl H2O, 20µl of the large fragment, 2µl of the short fragment, 1µl DpnI and 5µl 10x CutSmart buffer were incubated for 2 hours at 37°C
• Mixture was purified with isoprop, washed with ethanol and eluted in 10µl H2O
• 10µl Phuson Flash Master Mix was added and the followin protocol was run:

Cycles	temperature [°C]	Time [min:s]
1	98	0:10
5	98	0:01
	53	0:05
	72	3:00
1	72	10:00
1	10	inf

Unfortunately the efficiency of this was not high enough, as no colonies were visible on the plates after heat-shock transformation in BAP-I cells. The Amplification and CPEC will be repeated next week.