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.
Washington University, St.Louis, MO,USA. 63130.
Tsinghua U (B.S.)