help for long PCR product amplification - (May/31/2008 )

i try to amply 3kb fragment from genomic DNA of yeast, but the abundance of gene of interest is very low ,and everytime the PCR product i got is very weak.

i tried so many times but no results.

so pleast tell me what should i do?

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I have made good experience with finnzymes Phusion hot start enzyme. gave me considerable more product and virtually no background smear compared to conventional taq. processivity is also much higher, so you can use shorter extension times. optimal conditions are primers with tm of 65°C or higher, you can even run a two step protocol with annealing and extension at 72°C.

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Add an additional 2.5 mM MgCl2 - this will strengthen the annealing of your primers to the template. You can also reamplify your PCR product.

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If you are willing to read some, here are many links.
http://search.vadlo.com/b/q?sn=158621799&a...g+PCR&rel=0

1. I hope you are giving 3 minutes extension time.

2. An additional consideration in long PCR is not only success, but not introducing mutations. To that effect, you need to keep number of cycles as low as possible - unless it is some sort of diagnostic PCR.

..

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thanks for answer, but the most difficult thing is the abundance of the gene which has only one copy in genome .the abundance is very low , so the two steps protocol can be helpful?

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