[Arabidopsis] Could anyone help me on Arabidopsis genomic DNA cloning?

=?GB2312?B?88O0q8PA?= via arab-gen%40net.bio.net (by zhuchuanmei04 from gmail.com)
Mon Jun 29 19:21:15 EST 2009


Hi,
I am a graduate student in Washington University. I have trouble in cloning
a ~1.9 kb DNA fragment from Arabidopsis genomic DNA recently. The genomic
DNA was prepared by CTAB method and was dissolved in H2O.  For 25ul PCR
reaction, I use DNA ~500ng, MgCl2 1.5mM, dNTP 160uM, Taq 2.5U, primer each
0.2uM, buffer 1x. The PCR program was 94'  5min; 94'  45s, 54' 45s, 72'
2min, 20cycle; 72' 5min, 4' for ever.

I tried several times, including trying different concentration of DNA,
MgCl2... However, I never got the expected 1.9kb band? This was my first
time to clone gene from genomic DNA, I have no idea why it turns out to be
so difficult. Did you have any suggestions? Thanks in advance.

Best,

-- 
Chuanmei Zhu
DBBS(plant biology),
Washington University, St.Louis, MO,USA. 63130.

Tsinghua U (B.S.)


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