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Whole plasmid amplification by PCR


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#1 Chelo

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Posted 23 July 2013 - 06:48 AM

Dear forum members,
is it possible to amplify an entire plasmid by PCR so as to get enough material for mammalian cell transfection?
My specific problem is that my plasmid has a highly repetitive insert several kilobases in lenght so conventional amplification by cloning in bacteria is unpractical.
Is there any highly processive Taq-pol with proof-reading activity that I might use for this amplification?
Thanks in advance for your answers!

#2 bob1

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Posted 23 July 2013 - 12:37 PM

It is possible for sure, this is commonly used in "Qickchange" site directed mutagenesis and similar protocols. The difficulty is that as the plasmids get larger, it gets more and more difficult to amplify, the practical limit for these sorts of things is about 5 kbp due to the frequency of mutations that build up.

KOD and pfu polymerases are ones commonly used for this sort of thing.

#3 susnata

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Posted 02 August 2013 - 01:16 AM

i have used Pfu polymerase to amplify whole plasmid for SDM.. works fine






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