Engineering BioBrick vectors from BioBrick parts/Colony PCR

From OpenWetWare
Revision as of 07:51, 18 March 2008 by Reshma P. Shetty (talk | contribs) (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...)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to: navigation, search

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

  1. 95°C for 15 minutes
  2. 94°C for 30 seconds
  3. 62°C for 30 seconds
  4. 68°C for 3.5 minutes
  5. Repeat 2-4 39 times.
  6. 68°C for 20 mins
  7. 4°C forever