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Team:Freiburg/Notebook/Methoden
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Revision as of 18:29, 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 |
| Primer forward | 1,25 µl |
| Primer reverse | 1,25 µl |
| Template | 0,25 µl |
| Total | 25 µl |
run the following PCR program:
| step | 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 |
Repeat step 2. - 4. 15 times ans step 5. - 7. 30 times.
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
