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Revision as of 15:59, 14 September 2015
Colony PCR
Standard protocoll colony pcr
Colony PCR was tested by me (Stefan) for the insertion of an insert into pSB1C3 backbone. It is very importent to use primers with high binding affinities (such proposed by GATC) and with equal melting temperatures.
PCR - Mastermix
| µl | type |
|---|---|
| 2.5 | Taq Buffer |
| 1 | Primer1 |
| 1 | Primer2 |
| 2.5 | dNTPs |
| 0.125 | Taq Polymerase |
| Add to 25 | H2O |
- First step: 10 min 95 °C (Bacteria boiling)
- Then follow normal PCR protocoll but mind to use a extention temperature of 68 °C (1 min/kb).
Proceeding
Aliquote the prepared mastermix to the desired numbre of pcr tubes. Pick colonies with a pipet tip and bring them into the pcr tube by scratshing on the innter tube wall and mixing. Thereafter stap with the same pipet tip into a agar plate to recultivate the bycteria used in this colony pcr. (dont forget to mark the colonies on the new agar plate). Run the PCR and seperate bands by gel electrophoresis. Have Fun.
