"
Team:SDU-Denmark/labwork/Protocols/colpcr
From 2012.igem.org
| mRNA Isolation | PCR | Miniprep | Check Digest |
| 3A-Assembly | Colony-PCR | Transformation | Gel-electrophoresis |
| Reverse Transcriptase | Mutagenisis | PCR-,gel clean-up | Content |
Colony PCR
Transfer few cells from a colony to the bottom of a PCR tube. Add 100uL of sterilized water to the tube and microwave it at max effect for 2 mins to destroy the cells.Use 2uL of the colony DNA suspension in the following PCR mix.
5μl Dream Taq Buffer 1μl dNTP 1μl Primer1 (VF2) 1μl Primer2 (VR) 3μl 25mM MgCl2 1μl Dream Taq Polymerase 2μl Template suspension 36μl sterile destilled H2O
PCR program:
95°C for 2minutes30 cycles: 95°C for 1 minut melting temp -5°C for 30seconds 72°C for 1 minute/kb72°C for 10 minute 4°C on hold
