1
$\begingroup$

I'm using cDNA AFLP teq.. I extracted 670 bp band from polyacrylamide (urea) gel and I have amplified with PCR but on agarose gel it stops at nearly 350 bp.it happened for most of other bands before. I always extracted the precise bands. I repeated with both pre and selective primers with the same PCR program before done for amplification of digested cDNA and same size bands observed in agarose gel.I wonder maybe secondary structure causes losses in band size (before running in polyacrylamide samples treated in 94 degree for 5 min), but how I can recognize? i should notice 350 bp band i mentioned above is very sharp. what other thing I'm wondering is can dye uses in agarose gel alternate the DNA charge and reduces the speed of movement . The big matter is the rate of losses in band size almost is same for almost all of the bands for one primer. Is it happened for anyone before? can i trust on my extracted bands for doing cloning?

$\endgroup$
  • 1
    $\begingroup$ Urea-PAGE is a denaturing gel. The mobilities are likely to be different from the normal agarose gel. Can you put the gel pictures up? $\endgroup$ – WYSIWYG Nov 15 '14 at 8:12
  • $\begingroup$ I ran dna ladder in both acrylamide andagarose gel, if mobilities affected it should be true about the ladder! $\endgroup$ – sh.v Nov 15 '14 at 9:54
  • $\begingroup$ What ladder was it. Urea will denature dsDNA $\endgroup$ – WYSIWYG Nov 15 '14 at 10:24
  • $\begingroup$ I don't know realy. I always thought ladder is dsDNA. is there any ssDNA ladder. I should check it. I'll let you know soon. however thanks for your help. $\endgroup$ – sh.v Nov 17 '14 at 7:51
  • $\begingroup$ Thanks. with your guide I could find the problem, really there was no problem! DNA ladder I used was double strand DNA, and somewhere I read ladder's mobilization profile in denaturing gels differs depending on gel percent. I ran the PCR product of extracted band in polyacrylamide gel again. And I found what I expected, the same size -670bp. I think denaturing gel affected sample DNAs and ladder mobilization but because of their size and sequence (GC content for producing secondary structure)difference the influence was not same and it 's not predictable. $\endgroup$ – sh.v Nov 23 '14 at 5:08

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Browse other questions tagged or ask your own question.