Knight:Colony PCR protocol

From OpenWetWare

Revision as of 05:08, 24 September 2009 by Vaishnavi Ananth (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Solutions/reagents:

Parameters:

  • X - 1 min per kb of expected product.

Equipment:

  • Thermocycler
  • Electrophoretic unit
  • Reaction tubes

Steps:

  1. Reaction Mixture
    Use the following table as a checklist for preparing the reaction:

     PCR supermixVF2VRcolony template
    Colony PCR9 µl0.25 µl0.25 µl0.5 µl
  2. PCR conditions
    Program a standard thermocycler to run the reaction using the following parameters:
    Initial denaturation
    • Denature: 95°C, 15 mins
    Thermocycling
    • No. of cycles: 39
    • Denature: 94°C, 30 secs
    • Anneal: 56°C, 30 secs
    • Elongate: 68°C, X
    Elongation time : I typically round up for this step. i.e. For a 3.6kb construct, I used a 4 min elongation time. It seems to help to be a bit generous with the elongation time.
    Termination
    • Elongate: 60°C, 20 mins
    • Hold: 4°C, until removed from machine
  3. Perform agarose gel electrophoresis of appropriate quantity of PCR products mixed with ethidium bromide and visualize with UV transilluminator to confirm the presence of required product.

Personal tools