Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

PAGE problems

PAGE

  • Please log in to reply
4 replies to this topic

#1 hobglobin

hobglobin

    Growing old is mandatory, growing up is optional...

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,507 posts
94
Excellent

Posted 27 May 2013 - 07:47 AM

We're doing PAGE with PCR samples using a quite large chamber (16 cm running distance) and non-denaturating conditions. Gel concentrations differ depending on expected band sizes (we expect one to two alleles per PCR, band sizes are 100-300 bp). The gels are cooled during the runs.
Before PAGE the PCRs were optimised with gradient PCRs and different Mg2+ concentrations according to results visualised on standard agarose gels, where we got one or two clear bands as expected (rarely some unspecific bands and sometimes primer dimers). With PAGE we want now enhance resolution.

Anyway when doing PAGE we never have the expected one or two bands, but either four bands, many bands or even a smear-like pattern. The same occurs with the size ladder (we use standard size ladders such as NEB's low molecular weight marker). The latter is recognised by the manufacturers and they suggest to use RNA loading dyes (i.e. denaturating conditions).

The question is now, why these weird band patterns occur in the samples and how to avoid it? Is it a PCR problem with badly designed primers (we got them from literature and have not much additional sequence information for the loci, but anyway when I checked them, the primer design was okay for me) and many unspecific products which are not separated in an agarose run. Or is it a problem that the PAGE conditions are not denaturating? Or other possible reasons (e.g. DNA binding proteins that interfere?)?
Thanks for any input and ideas...

Edited by hobglobin, 27 May 2013 - 09:22 AM.

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,706 posts
123
Excellent

Posted 28 May 2013 - 04:00 AM

have you monitored the temperature of the gel during the run? you may be melting the pcr products while they are traveling through the gel.

you can try a denaturing (urea) gel to determine if your pcr product is specific.
talent does what it can
genius does what it must
i do what i get paid to do

#3 hobglobin

hobglobin

    Growing old is mandatory, growing up is optional...

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,507 posts
94
Excellent

Posted 28 May 2013 - 06:55 AM

have you monitored the temperature of the gel during the run? you may be melting the pcr products while they are traveling through the gel.

you can try a denaturing (urea) gel to determine if your pcr product is specific.

didn't do that so far, but I'll check it, somewhere we had an infrared thermometer...
Is it enough to use a denaturing loading buffer or heating the sample or has to be the whole gel denaturing?
thanks.
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

#4 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,706 posts
123
Excellent

Posted 29 May 2013 - 06:21 AM

if the whole gel isn't denaturing then you may find the sample renaturing during the run.
talent does what it can
genius does what it must
i do what i get paid to do

#5 hobglobin

hobglobin

    Growing old is mandatory, growing up is optional...

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,507 posts
94
Excellent

Posted 29 May 2013 - 07:30 AM

if the whole gel isn't denaturing then you may find the sample renaturing during the run.

okay then we try it with denaturating gels...
The temperature seems no problem as it is continuously around 25 degrees...until 50 degrees it should be no problem or even helpful to decrease running time...
Thanks mdfenko
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.