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Team:SDU-Denmark/labwork/Protocols/PCR
From 2012.igem.org
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1. Start 95°C 2min </br> | 1. Start 95°C 2min </br> | ||
2. Denaturing 95°C 1min </br> | 2. Denaturing 95°C 1min </br> | ||
Revision as of 20:18, 15 September 2012
| mRNA Isolation | PCR | Miniprep | Check Digest |
| Content | Content | Content | Content |
| Content | Content | Content | 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
