PCR amp. - sequencing problem - (Jul/16/2006 )
Hi there,
Currently I am trying to amplify DNA fragments with one set of primers, then using the forward primer from the same set to conduct sequencing PCR. before I do sequencing, I do gel purification. However, this has given me no sequencing results. My hypothesis:
- the DNA ends have degraded, so the same primers cannot attach
- some adduct that passes through the purification process attaches to the ends, preventing the primers from attaching
Are these possible reasons? Has anyone encountered the same problem? If I use a different forward primer for my sequencing reaction, how far away should it be from the PCR primer?
Thank you!
Do you need to gel purify? Regular PCR purifiation you do not expose your DNA to UV therefor you have smaller chance of degradation.
What's the Tm of your primer? Have you tried your reverse primer?
What do you mean with "no sequencing results"? An empty electropherogram, or a very unclear signal (as for instance frameshifts would cause)? (in the first case your primer probably doesn't anneal, in the second case it would anneal on several sites).
Hi,
I use nested primers (>10 nt from the 3' end of outer primer) for sequencing but do have a friend who did the same as your method without problem. Things to consider during primer design would be the Tm and also the dimers.
Since you have a product to visualize under UV in agarose gel (I assumed), then the primers worked fine.
Well, the problem probably lies in the sequencing part. Try as Vairus suggested, i.e. use the reverse primer. Optimize the PCR product conc. used in sequencing, e.g. too much will cause depletion of dyes in the early stage of cycle sequencing. Try to increase no. of cycle or the primer concentration. Check if Big Dye is still ok.
Good luck.
What's the Tm of your primer? Have you tried your reverse primer?
What do you mean with "no sequencing results"? An empty electropherogram, or a very unclear signal (as for instance frameshifts would cause)? (in the first case your primer probably doesn't anneal, in the second case it would anneal on several sites).
Thanks for your response. We have to do gel purification because of the nature of our sample...one chromosome has a neo insertion, but we are primarily interested in the chromosome without it. Therefore, we have to gel purify or else sequencing would occur on both alleles. The Tm of the primer is lower than that of the sequencing PCR settings, therefore it should "melt" properly, right? We have not tried the reverse primer yet; we might carry on this suggestion. Thanks!