Long Pcr...Ecperience and Suggestions (2.5 KB from Plasmid + linker)
snyder.9 at osu.edu
Sun Jun 2 20:48:47 EST 1996
In article <31B4254B.3116 at odyssee.net>, dellaire at ODYSSEE.NET (Graham
> Hello all,
> I am going to do some PCR cloning and I need to amplify a 2.5 kb piece
> of DNA from one plasmid to clone into another. I want to use one
> specific 20 mer and another 30mer with 20 bases specific and 10 more
> that contain a specific AscI site for cloning (gc rich). I am thinking
> about using Vent pol. but I have never amplified anything over 1Kb...
> Any suggestions on primer design... should I have the AscI site internal
> or is it better on the 5' end??
> What are the pitfalls I should watch out for... besides standardizing my
> Mg conc and nucleotides?? I heard that the extension times should be
> increased... aproximately 1 minute/Kb....?
> Thanks for any and all suggestions,
> Graham Dellaire
> McGill Dept. of Medicine
> Div. Of Experimental Medicine
> dellaire at odyssee.net
I have amplified up to 5 kb from plasmid with no special changes made to
the reaction. just regular taq. our reaction is pretty generous in GCAT
to begin with and we regularly use a 3 minute extension in our water
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