Smeared Bands in PCR

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Fri Dec 13 00:06:48 EST 1996


There are lots of reasons for getting smears (and different kinds of
smears can mean different things), here is an incomplete list:

1) Too much template.  This can look like a smear at the top
2) overcycling, if your dNTP's are used up, incomplete extentions can
form all kinds of complexes.
3) extension times that are/become too short.  Same reasoning,
incomplete extentions by polymerase that is inadequate at extending
all the available template.
4) Annealing temp is too low.  In this case, you amp up sequences
other than the ones you want.
5) No Hot start. Again, the primers hit and extend on too many
undesired sequences while your rxn is sitting on your bench or ramping
up to the first denature
6) Your template some sort of contaminant that is producing the
abberant behavior.  (Numerous, sometimes very odd, sources of PCR
inhibitors have been reported).
7) Reagents are degraded and/or contaminated.  self explanatory
8) None/all/some of the above

Hope this is of some use					SVEN


In article <32AE53A1 at SmtpOut.em.cdc.gov>, tar9 at NIOBBS1.EM.CDC.GOV ("Reid, Thomas M.") writes:
> I am having problems with smeared bands when analyzing PCR products on 
> agarose gels.  I am amplifying a 600 bp segment of a 25 Mbp fungal genome. 
>  The smearing does not always occur although it seems to be somewhat 
> polymerase-specific.  The smearing is above the major band so I don't think 
> it's an exonuclease problem.
> 
> I would appreciate any advice, thoughts, etc.
> 
> Tom Reid



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