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PCR Walking got same size bonds for different library


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#1 ophrys

ophrys

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Posted 09 December 2009 - 04:57 AM

Hi all,
I am trying to get the promoter sequence of my gene. I used the method of Genome Walker from Clontech. Well, it works somehow, i got one bonds after two rounds of PCR, and i've clone and sequenced it, it is the up stream sequence of my gene. However, the size of this fragments is just about 500bp, I designed different primer, try to get longer upstream fragments, it didn't work.

Then, i tried digest the genome with other eight different enzyme, and performed same procedure, at the end, i got one bond for all the these eight libraries, but the bonds are same size as i got before.

I also tried to amplify different gene with same library, it gave me very good results, the size of the amplified bonds were different from these
libraries. (from 0.5 - 1kb ), and sequence matched well. It means the enzyme cut genome well, and adaptor was ligated well.


I am really confused about this results. It impossible that all enzyme cut same site of the genome, why different library gave same fragment? (there are slitty differences, some has two bonds, some only has one, but the biggest bond was always same, around 0.5 kb).
Anybody has a clue?

From the sequence i've got, it seems the upstream sequence of this gene has lot of AT rich sequence, will this affect the size of PCR amplification?

BTW, The polymerase i used was from Clontech GC Advantage 2 polymerase.


Thanks a lot.

Steven




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