"

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
Water, nuclease-free	up to 20 µl
Total vloume	20 µl

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: