sequence is blurry

[David Micklem]

In article <538vh2$cev at bertrand.ccs.carleton.ca>,
jwhitewa at chat.carleton.ca (Jacqui Whiteway) wrote:

>gc (genecutl at mendel.berkeley.edu) wrote:
>> I've been having trouble reading my sequencing gels because the
>> bands tend to get blurry after about 60-100 bases from the bottom
>> and I can't tell if I'm looking at single bands or doublets.  I've 
>> been running 5% acrylamide gels at 35-40 watts.  The buffer is 
>> 1xTBE, except that I add sodium acetate to 1M in the bottom buffer
>> after the gel has run about half way.  Any suggestions on sharpening 
>> my bands would be grealy appreciated.
>
>> --gc
>> TheRoadToNowhere
>> http://sp1.berkeley.edu/

Could you be running your gel too hot?  I use a stick-on thermometer (LCD)
to measure the temperature near the top of the plate - it shouldn't go
above 50-55C..

The power you run your gel at is not very useful unless you also tell us
the dimensions of your plates. (IIRC the temperature vs power is related
to the _volume_ of the gel).
For a roughly 50ml 5% gel I run at 35-40W, but I don't use 1M NaAc.  (I
tried it, but it made my lanes spread out sideways off the edge of the
gel! Anyway, we have an excellent set-up for pouring buffer-gradient
gels...)

David

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D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
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