Long Pcr...Ecperience and Suggestions (2.5 KB from Plasmid + linker)
dellaire at ODYSSEE.NET
Tue Jun 4 07:12:55 EST 1996
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,
McGill Dept. of Medicine
Div. Of Experimental Medicine
dellaire at odyssee.net
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