etbr in gels / interlanular smiling

suter at de.dbp.MPG.MPIZ-KOELN.VAX suter at de.dbp.MPG.MPIZ-KOELN.VAX
Tue Aug 4 12:55:17 EST 1992


<Subj:	Re: Smiles on Southerns
<
<If you run a large amount of DNA in a lane of a gel containing ethidium bromide
<the DNA tends to bind the dye. If the ethidium bromide is limiting then there
<may not be sufficient to saturate the DNA. At the _edges_ of the lane there
<will be more ethidium bromide available (it will be able to diffuse in from the
<spacer agarose between lanes) and so the DNA at the edges will tend to have
<more ethidium bromide bound. The efect of this will be to retard the DNA at the
<lane edges, hence smiling.
<
<This is easily tested. If you load decreasing amounts of DNA the smiling will
<reduce as the amount of ethidium bromide available will not be limiting for
<lesser amounts of DNA. Increasing the ethidium bromide concentration will also
<counter the effect.
<
<If you still need convincing then set up two gels that differ only by one
<containing ethidium bromide and the other not. Run identical samples of DNA
<(using amounts known to normally induce smiling) on both gels for the same
<amount of time. After electrophoresis stain the gel which had not contained
<ethidium bromide. Look, no smiles!
<
<Hope that this helps.
> Dr Raymond Dalgleish

don't think it does. i've had these smiles in both
EtBr containing gels and gels without EtBr. It does have to do with
overloading though. I think it helps a lot to load the dna (in a cleaner form)
in the slots and to leave the gel with dna for 10 to 20 minutes without 
current on it. Then switch on the gel and let it run at a very low current
and voltage. also: put a lot of buffer on the gel (1 cm). after 30 minutes,
i then turn up the voltage to 50V, 180mA (but this depends on your tank size).

i guess this way the dna sample has a chance to equilabrate to the electr.
buffer. 

clemens
----------------------------------> cut here <----------------------------------
Clemens Suter-Crazzolara, PhD
Max-Planck-Institut fuer Zuechtungsforschung
Abteilung Genetische Grundlagen der Zuechtungsforschung
Carl-von-Linne Weg 10
5000 Koeln 30
Tel. xx49-221-5062.221           fax. xx49-221-5062.21
suter at vax.mpiz-koeln.mpg.dbp.de
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