Rao:ColPCR

From OpenWetWare

Revision as of 14:16, 28 January 2007 by LonChubiz (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Background

Colony PCR verification, or whole colony PCR, is a now standard method to verify the modification of plasmid and/or chromosomal DNA replacing older methods such as radio-labeled hybridization. The method works by using the cellular DNA (chromosomal and/or plasmid) of selected colonies as template and using specific primers to generate PCR products to verify the presence of DNA manipulations. The selection of appropriate primers is essential for this technique to work well and to provide useful information to the investigator. The primary advantage of this technique is speed; the investigator can screen 10's to sometimes 100's of colonies quickly and with adequate precision and accuracy.

You'll Need

  • PCR tubes (0.2 mL) - Strips of 8 are nice.
  • Pos/Neg control vector
  • Set of primers that will give you a UNIQUE product for your DNA manipulation.
    • Don't select primers that will tell you just whether your insert/new DNA is present. Use something that will tell you about its location. If you need help with this... ask someone.
  • 2X Taq mastermix (Promega, Invitrogen, etc. make good ones)
  • Purified water
  • 1 plate for each appropriate anti-biotic you have selected with.
  • Tips and your P20 pipetman (20-μL pipeter for all you non-Rainin users)

Procedure

  • Make sure you have enough tubes for at least 3 or 4 verifications per plate + 1 for each control
  • Fill all the PCR tubes with 14.5-μL including your controls
    • MAKE SURE YOU LABEL YOUR TUBES NOW!
  • On the plate make rows/regions/number squares/whatever will keep track of your selected colonies.
Personal tools