Update of PCR steps for the pKO3 (pKOV) gene-replacement protocol

The following exerpts are from an article in preparation based on Dereth Phillips' Harvard Thesis:

PCR conditions.  The following PCR protocol was  modified from Cheng et al., PNAS 91, 5695-5699 1994. The concentration of reagents was as follows: 0.4 micromolar each primer, 85 mM KOAc, 25 mM Tricine pH 8.7, 8% glycerol, 1% DMSO, 0.2 mM dNTPs, and 1.1 mM Mg(OAc)2.  The template was added to the mixture in the form of either genomic DNA or whole bacterial cells.  The polymerase mixture was made by mixing rTth (Perkin  Elmer) and Vent (NEB) polymerases in a unit ratio of  rTth:Vent of 12.5:1.  All reactions were performed in a 50 microliter volume.  After a hot start at 94 degrees C the reactions were cycled 30 times at 94 degrees C for 10 seconds and 65 degrees C for 3 minutes.  A final hold of 10 minutes at 72 degrees C was added to allow complete extension of the products.  All primers contained a single phosphorothioate linkage between their 3' most bases.

Creation of deletion mutants.  Deletion constructs were created essentially as described in (Link et al., 1997) with the following modifications.  In our procedure the sequence tag used to replace the deleted region was the 33 bp sequence we refer to as "C22":  GTTATAAATTTGGAGTGTGAAGGTTATTGCGTG.    Deletion primers were picked using a program written by Keith Robison.  All such primers were designed to have a Tm between 65 and 70 degreesC and contained a single phosphorothioate linkage between the 3' most bases.   The phosphorothioate linkage  has been shown to improve amplification by preventing digestion by the exonuclease activity of vent polymerase (Skerra, 1992).  A modified form of the pKO3 gene-replacement vector, pKOV, was used. (Link et al., 1997)

[note: Although the literature indicates that the phosphorothioate (sulfur) linkage protects the primer from digestion by the exo activity of the Vent polymerase, it may not be absolutely necessary in our protocol.]

The following was an exerpt from our lab pKO3/V manual:
 [note: the concentrations in the recipes are working concentrations, not final concentrations]

 50 microliter reaction to amplify flanks
 2 microliters primer#1 (10 pmole/microliter stock, i.e. 10 micromolar)
 2 microliters  primer#2 (10 pmole/microliter stock, i.e. 10 micromolar)
 1 microliter genomic DNA (approx 1 microgram/ ml stock)
 10 microliters 5x long PCR buffer (Cheng et al. recipe)
 5 microliters dNTP's (2 mM stock)
 2.2 microliters Mg(OAc)2  (25 mM stock) (2.4 microliters may make for a better PCR but  may be more mutagenic)
 26.8 microliters H2O

Hot start (see MJR-PTC100 program below) then add:
 1 microliter of polymerase mix
       (10:1 vol:vol mix of rTth (Perkin Elmer) 2.5U/microliter and Vent (NEB) 2U/microliter)

To make the crossover product, you must mix the flanks in a second round of PCR.  For the template DNA, take 1 microliter of the reaction product from each of the N-flank reactions and mix it with 1 microliter of the the reaction product from the corresponding C-flank reactions.  Use the outer primers (No and Co) to amplify the crossover product.  Perform these reactions in duplicate.

 50 microliter reaction for crossover PCR
 2 microliters  primer No (10 pmoles/microliter stock)
 2 microliters  primer Co (10 pmoles/microliter stock)
 1 microliter  from N-flank reaction above
 1 microliter from C-flank reaction above
 10 microliters 5x long PCR buffer
 5 microliters  dNTP's (2 mM stock)
 2.2 microliters  Mg(OAc)2 (25 mM stock)
 25.8 microliters H2O

Hot start then add:
 1 microliters polymerase mix
       (10:1 vol:vol mix of rTth (Perkin Elmer) 2.5U/microliter and Vent (NEB) 2U/microliter)

PCR program for MJ Research, Inc.  PTC-100:

  94 degrees - 1 minute
  88 degrees - 4 minutes (load during this time for hot start, press pause if you need more time)
  30 cycles of:
      94 degrees - 10 seconds
      65 degrees - 3 minutes
  final extension:
  72 degrees - 10 minutes
  3 degrees - 99h or "hold"

This page was updated 7-1-2000 by Dereth Phillips and George Church