PCR 4.5kb plasmid

norm eberhardt eberhard at Mayo.EDU
Thu Mar 4 08:11:24 EST 1993

In article <1993Mar3.064521.17305 at Princeton.EDU>, angelo at phoenix.Princeton.EDU (Angelo Gunasekera) writes:
|> In protein engineering meeting in SanDiego, I seem to remember seeing a
|> poster (or a paper) discussing about PCR'ing a whole plasmid to
|> incoorporate a sequence into the plasmid (by add-on PCR with 2 primers T
|> and B strands which prime in the same region having approximately
|> 40bases in each of the primers as added-on's).  I think they just used
|> ligase after their PCR and transformed into E.coli cells to get very
|> good incorporation of the sequence of their interest.
|> I am wondering someone in the bionet could direct me to get that paper
|> or help me getting a similar protocol.
|> Thanks in advance
|> Angelo 
	We do this kind of operation routinely to do either add-on, deletion, or
site-specific mutagenesis.  The article which forms the basis of our mutagenesis

Hemsley A, Arnheim N, Toney MD, Cortopassi G and Galas DJ.  A simple method for
site-directed mutagenesis using the polymerase chain reaction.  Nucl Acids Res
17: 6545-6551 (1989).

	We have modified the reaction conditions very little; however, the 
following tips will help to improve efficiency:

1.  Use ca. 5 ng plasmid template.
2.  Use Pfu polymerase  (Statagene) to avoid unwanted mutations.
3.  Pfu avoids necessity to Klenow-treat the PCR fragments, since it does not
    add As.
4.  Gel isolate and "Geneclean" the PCR products to remove supercoiled template
    DNA which results in high backgrounds.
5.  5'-Phosphorylate the primers, not the PCR products, to enhance ligation

	We have done ca. 50 mutations of various DNAs using this technique.  We
are interested in 250-500bp fragments, which we then sublcone into fresh
expression vectors to avoid the possibility of incorporating a mutation into
another part of the vector which affects the kinds of studies we are doing.  We
usually do this kind of mutagenesis in pUC vectors (ca. 3.2kb with inserts)
but have successfully ampflified vectors up to 6.5kb using this technique.

				Hope this helps,
				norman eberhardt
				eberhardt at mayo.edu

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