RNA electrophoresis help

Nick Theodorakis nick_theodorakis at hotmail.com
Tue Apr 11 09:24:03 EST 2006


John Frank wrote:
> i recently ran a RNA agarose electrophoresis (in denaturant conditions/formaldehyde) with ethidium bromide in the gel. since not even the markers were visible under the UV, i was wondering if anyone is aware of any possible interactions of the MOPS/formaldehyde/formamide mixture with the ethidium bromide (i am asking this because after seeing some protocols describing this procedure, i noticed people tend to stain with EtBr afterwards, as opposed to including it in the gel). thanks in advance :)
>

There is a lot of background fluorescence if you put ethidium in a
formaldehyde gel. I put it in the loading buffer only, and the
background is nice and dark.  Also, you can monitor the gel as you run
it.

Here is my recipe:

Northern Sample Buffer: (50 ml)
  5 ml 10 X MOPS
  5 ml Glycerol
  25 ml formamide
  8 ml 37% formaldehyde
  0.2 ml 10 mg/ml ethidium bromide
  0.03 g bromophenol blue (or add 0.3 ml of 1% bromophenol blue)
  add water to final volume 50 ml
  aliquot and freeze

Add 2-4 volumes of buffer to RNA sample, heat to 65 C for 5 min before
loading

Nick

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Nick Theodorakis
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