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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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Sequencing PCR problem
Posted by: dadashi_2000 (IP Hidden, New member, 2)
Date: July 18, 2007 11:40PM
Dear friends,
I am a new member of this forum. Actually i was thinking about problem with my sequencing PCR. I did successfully this simple experiment many times with various plasmids extracted from E. coli. Recently I need to sequence the new cloned gene but I have NNNNNNN in my sequencing sheet. I changed everything that you may think! but the results are the same. It would be really appreciated if somebody shears his/ her experiences with me. Thank you in advance, M. Dadashi
Re: Sequencing PCR problem
Posted by: slimshaddy (IP Hidden, New member, 2)
Date: July 19, 2007 01:30AM
Check ur primers of seq pcr. Also check the ur adding the bigdye properly. Hope u wash the seq plate perfectly and not losing pcr product.
Re: Sequencing PCR problem
Posted by: dadashi_2000 (IP Hidden, New member, 2)
Date: July 19, 2007 04:56AM
Thanks for your reply,
I think there is no problem with primers because when i use the same primer with control DNA, the result is OK! BigDye or our premix solution is the last component of reaction mixture and after mixing, I spin down it properly. About sequencing plate, other people get good results with the same ABI system. Is there any probable role for RNA contamination in PCR? I don't know well , but now i am purifying the plasmid after extraction by RNase and PEG- NaCl method.... Thanks a lot, Dadashi
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