amplification of 13Kb DNA fragment by PCR

San-pin Wu wusp at GATE.SINICA.EDU.TW
Mon Aug 14 23:54:22 EST 1995


    First fo all, thank your for your reply for my question. The procedure
what I treat to my PCR mixture is that phenol(pH8.0)/chlorofrom extraction
then EtOH precipitation. After that, the pellet was dissloved in 20uL TE
buffer and loaded into 0.8% agarose gel. The pattern what I saw on the gel
was that My DNA disspeared and the whole lane smear! If the PCR mixture
was loaded into gel without any concentrated process, the bend was very
sharp. So I turned to Centricon(Amicon, Inc.) to try to elute out those
useless components in the PCR mixture. But the result was the same with
EtOH precipitation. On the other hand, I tried to cut the smeared agarose
gel pieces which might contain my DNA and reextracted them again. The
result is that I get my DNA back but with very low recovery! I really do
not know what's going on with my PCR mixture?! 

     Thank you for your help again.

                                             Steve Wu
                                             Institute of Botany
                                             Academia Sinica
                                             Taipei, Taiwan
                                             E-mail:wusp at gate.sinica.edu.tw





More information about the Methods mailing list