Sequencing gel smears

Robert Hartley rh at mblab.gla.ac.uk
Thu Aug 3 11:12:16 EST 2000


In article <l03130300b5af2df0673a@[130.237.135.69]>,
Neil.Portwood at cck.ki.se (Neil Portwood) wrote:

Sorry folks for not following ettiquette and posting a reply at the top.

BUT

I presume you mean that you are doing RT then PCR then running on a
urea/acrylamide gel. IE a sequencing gel.

I also assume that you are expecting a couple of bands but you get a
couple of bands but there is a big dark smear from the exposure throughout
the gel.

I wouldn't have thought this was the answer but when some folk use
OloigodT in differential display they get this effect. (also heavier
exposure at the top of the lane) If you us a RT promer that is oligo dT
with a single base anchor this might work

IE TTTTTTTTTTTTTTTTTc or g or A (less so) then this might help.

Oonly a guess though.

cheers
Bob; Rainy Scotland and I don't have a coat. (morral of the story? where
did all the weather forcasters get their degree? in a crisp packet?) :-)


> Hi,
> Are there any kind souls who can help? I'm performing fragment quantitation
> of 32P-labelled multiplex PCR products on denaturing gels. Unfortunately,
> in addition to the expected products are present two to three lines of
> smear which variously migrate as a smile or a grouch. This symmetry, and
> the fact that each smear appears to be specific to one or other of the
> expected products, suggests that the smears represent partial renatured
> forms of the expected products, their mobility being determined by
> differential thermal properties at the center versus the sides of the gel
> during the run (they are also not visible on agarose gels). The smears make
> quantitation impossible: they sometimes co-migrate with the expected
> products in, say, the center of the gel, and confound the analysis by
> dividing signal between a specific band and its 'smear' in different
> proportions, depending upon where on the gel a sample is loaded, eg in the
> center or at the sides. Worse, in lanes where only small amouts of total
> PCR product are loaded, only the specific products are visible (ie no
> smears), whereas smearing appears where the total amount of loaded product
> is more. The PCR (5 microliter volume) is running for only 28 cycles, so
> from agarose gel band intensities I'm pretty sure this isn't a straight
> overloading problem. I've tried various TBE concentrations between 0.75x to
> 2x (both gel and buffer), but with little effect. Could this be a buffer
> exhaustion problem, in which case could running gradient gels cure the
> smears? Just in case anyone feels it's relevant, the TBE and Sangar reagent
> are prepared fresh for every run, I do not routinely degass the Sangar and
> the acrylamide is an Ultrapure solution from Life Technologies. Any
> suggestions are welcome!
> 
> Dr. Neil Portwood,
> Radiumhemmets Research Laboratory,
> Department of Oncology-Pathology,
> Karolinska Institute,
> CancerCentrum Karolinska R8, 3rd Floor,
> Karolinska Hospital,
> 171 76 Stockholm,
> Sweden.
> 
> Tel: 46-8-5177 5408/46-8-5177 5460
> Fax: 46-8-33 90 31
> e-mail: Neil.Portwood at cck.ki.se
> 
> 
> 
> ---






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