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Team:SDU-Denmark/labwork/Protocols/colpcr
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| - | 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. | + | 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 template suspension in the following PCR mix. |
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Revision as of 23:25, 24 September 2012
| 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 template suspension in the following PCR mix.
PCR program: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
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
