2 replies to this topic

[naturalman]

Hi, Dear friends,
I am frustrated by my PCR experiment. I wanted to clone a gene from the cDNA samples. Probably due to the degradation of the template DNA, the amplified band was quite weak (Lane 1). So I tried to purify the interest DNA from the gel and the second PCR was carried out with the template of the purified DNA. However, the product seemed to be too much and smeared along the lane after electrophoresis (Lane 2). This happened when I did PCR using directly the PCR product as template (Lane 3).
Will you please explain the result for me and tell me how to overcome it?
Thanks in advance![attachment=3389:why smear.jpg][attachment=3389:why smear.jpg]

[phage434]

This is very common when using the same primers in a second round of PCR.  If possible, use a different primer for the second round (or even change just one of the two primers).

[naturalman]

phage434, on 20 September 2011 - 04:29 AM, said:

This is very common when using the same primers in a second round of PCR.  If possible, use a different primer for the second round (or even change just one of the two primers).

Thank you very much for your advice. I will try as you said. However, in fact, I just want to amplify the DNA fragment more.