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Team:Freiburg/Notebook/Methoden
From 2012.igem.org
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This step is necessary to add the the necessary restriction sites to the Direpeats. | This step is necessary to add the the necessary restriction sites to the Direpeats. | ||
Revision as of 17:14, 25 September 2012
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
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:
