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Team:SDU-Denmark/labwork/Protocols/PCR
From 2012.igem.org
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Revision as of 22:08, 17 September 2012
| mRNA Isolation | PCR | Miniprep | Check Digest |
| 3A-Assembly | Colony-PCR | Transformation | Gel-electrophoresis |
| Reverse Transcriptase | Mutagenisis | PCR-,gel clean-up | Content |
Polymerase Chain Reaction
Proof-reading PCR using Phusion Hot Start II
PCR program:
95°C for 30 seconds25-35 cycles: 98°C for 30 seconds Annealing temperature (5°C below primer melting temperature) 72°C for 15-30s/kb72°C for 5-10 minutes 4°C on hold
Non-proof-reading PCR using Taq Polymerase
5μl Dream Taq Buffer 5μl dNTP 1μl VF2 1μl VR Udtag 10μl fra primer stock og fortynd i 90μl oprenset vand 1,5μl Dream Taq Polymerase 1μl Template H2O up to 50μl PCR-PROGRAM:1. Start 95°C 2min 2. Denaturing 95°C 1min 3. Annealing 55°C 30sek 4. Elonging 72°C 1min (1kb/min) 5. GO TO step 2, x 29 cycles 6. End 72°C 5min 7. Hold 4°C infinite
