Long Pcr...Ecperience and Suggestions (2.5 KB from Plasmid + linker)

[Graham Dellaire]

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