Template:IONIS Paris/Notebook/03-07-15

PCR pDawn BioBricks

PCR pDawn III (2)

Aim: first step for pDawn amplification and mutagenesis

Mix PCR preparation:

	pDawn III
MQ water	25,5 µL
Q5 Buffer	10 µL
High GC content Buffer	10 µL
dNTPs 10µM	1 µL
Primer Fwd 50µM	1µL pDawn Fwd III
Primer Rev 50µM	1µL pDawn Rev III
DNA	1µL pDawn
Taq Pol	0.5 µL

PCR cycle – IGEM TAQ 2

	T°C	Time	Cycle
Initial denaturation	95	30 sec	1
Denaturation	95	30 sec	17
Annealing	60	30 sec
Extension	72	2 min
Final extension	72	5 min	1
Hold	4	Infinite

Electrophoresis

Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of pDawn III

Expected results

Results

No amplification

3 July 15