amplification of 13Kb DNA fragment by PCR (fwd)

San-pin Wu wusp at GATE.SINICA.EDU.TW
Thu Aug 10 21:55:53 EST 1995


    I have amplified a 13Kb DNA fragment from spinach chloroplast by the 
XL PCR Kit of PERKIN ELMER recently. But the yield is very low--about 
2ug/100ul/reaction and it seems hard to purify and concentrate the PCR 
product. I had tried to wash and concentrate them by Centricon-100 to 20ul 
and load into low-melting gel to try to get the fragment. The pattern 
what I see on the gel is that there is still a lot of primer exist and 
the band what I want disappeared!?! But I can see it on the 0.8% agarose 
gel while I diluted the PCR product mixture 5X with TE buffer!! It's 
really a strange thing. 

     Now I have DNA but can not purify and concentrate them!

     Welcom any comment or suggestion.

           
  Steve Wu
  Institute of Botany, Academia Sinica, Taiwan
  wusp at gate.sinica.edu.tw
  




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