On Jun 29, 8:21 pm, 竺传美 <zhuchuanme... from gmail.com> wrote:
> 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.)
First at all, I would recommend to use 35 or 40 PCR cycles instead 20.
Second, I will use a hot start Taq an use 10 min. of initial
denaturation at 95 ºC, then the following denaturation steps should be
1 min. at 95 ºC. As tip I recommend to pipette several times the DNA
before add it to the PCR reaction.
Hope this help!
Adrian Moreno, M.Sc.
Centro de Biotecnologia Vegetal
Universidad Andres Bello
Republica 217, Santiago 837-0146
CHILE
