Template:IONIS Paris/Notebook/06-05-15

PCR VVD BioBricks

PCR.1 VVD

Aim: first step for VVD amplification and mutagenesis

dNTP preparation (4µL):
1 µl dATP 100 mM
1 µl dCTP 100 mM
1 µl dTTP 100 mM
1 µl dGTP 100 mM
Store at -20°C

PCR cycle – IGEM TAQ

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

Mix PCR preparation without primers, plasmid and Taq

MQ water	182,5 µl
Mg2+ 50 mM	7,5 µl
Buffer RB	25 µl
dNTPs	2,5 µl

Mix preparation for 5 tubes: 43,5 µl / tube

6 May 15