"

From 2012.igem.org

First Extension PCR (Toolkit)

This step is necessary to add the the necessary restriction sites to the Direpeats.
Pipette the following volumes into a PCR- tube:

Component	Volume
Water, nuclease-free	15,75 µl
DMSO	0,75 µl
dNTPs	0,5 µl
Phusion Buffer	5,0 µl
Phusion Polymerase	0,25 µl
Primer forward	1,25 µl
Primer reverse	1,25 µl
Template	0,25 µl
Total	25 µl

run the following PCR program:

	temperatur	time
1.	98 °C	30 s
2.	98 °C	8 s
3.	68 °C	15 s
4.	72 °C	4 s
5.	98 °C	8 s
6.	70 °C	15 s
7.	72 °C	5 s
8.	72 °C	5 min
9.	4 °C	store

Second Extension PCR (Toolkit)

This step is necessary to add enzyme- binding –sites to the outer restriction sites. Use the product of the first extension- PCR as template.
Pipette the following volumes into a PCR- tube:

PCR Purification

bla

Gel Run

bla

Ligation

bla

Colony PCR

bla

Mini-Prep

bla