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

[Pam Snyder]

In article <31B4254B.3116 at odyssee.net>, dellaire at ODYSSEE.NET (Graham
Dellaire) wrote:

> 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
cooled thermocyclers