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Long Range PCR

Long Range PCR - PCR - Polymerase Chain Reaction Forum

Long Range PCR - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 12-08-2009, 09:40 PM
Pipette Filler
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Default Long Range PCR



Hi.

I am to PRC out an 8kbp exon divided into 10 parts. Standard PCR was giving messy sequence so my PI suggested using Expand Long range PCR. This is my first experience in a lab and this technique is a new to me. I followed the protocol that came with it and used the Forward and Reverse Primers as such: 1F,10R ; 1F,5R & 5F,10R; 1F,3R & 4F,6R & 7F,10R (Buffer with Mg and Buffer without Mg at 55C, 60C and 65C).
Protocol:

dH2O: 24.3
Buffer: 10
dNTP: 2.5
F: 2.5
R: 2.5
DMSO: 2.5
Enzyme: 0.7
Template: 5
Total vol : 50microliters

I finally got a strong band for 1F,3R. I still have to PCR the rest of the exon. Any suggestions? Am I doing something wrong?

There is a vial in there labeled 25mM MgCl2 which seems to have no use in the protocol. Could you tell me what that is for?

If I run out of the enzyme, is it okay to use Taq? Has anyone had success with that? I have ordered 2 kits only because the amount of the enzyme is low and I ran out while trying to get bands, while the other solutions are in plenty.

I would really appreciate any help, any advice.

Thanks
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