Engineering BioBrick vectors from BioBrick parts/Colony PCR: Difference between revisions
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(New page: ==Materials== *[http://www.invitrogen.com/content/sfs/manuals/11306016.pdf PCR SuperMix High Fidelity] *VF2 primer (<code>5'-TGCCACCTGACGTCTAAGAA-3'</code>) *VR primer (<code>5'-ATTACCGCC...) |
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Revision as of 08:51, 18 March 2008
Materials
- PCR SuperMix High Fidelity
- VF2 primer (
5'-TGCCACCTGACGTCTAAGAA-3') - VR primer (
5'-ATTACCGCCTTTGAGTGAGC-3') - Deionized, sterile H2O
- strip of PCR tubes
Equipment
- DNA Engine OPTICON\texttrademark from MJ Research, Inc. (now Bio-Rad Laboratories, Inc., Hercules, CA).
Procedure
PCR mix
- 9 μL PCR SuperMix High Fidelity
- 6.25 pmoles VF2 primer
- 6.25 pmoles VR primer
- 1 μL colony suspension
- dilute 1 colony in 100 μL water
PCR conditions
- 95°C for 15 minutes
- 94°C for 30 seconds
- 62°C for 30 seconds
- 68°C for 3.5 minutes
- Repeat 2-4 39 times.
- 68°C for 20 mins
- 4°C forever
