Request for a 6x gel loading buffer receipt

Chris Boyd chrisb at hgu.mrc.ac.uk
Mon Aug 2 02:22:33 EST 1999


Frank O. Fackelmayer (Frank.Fackelmayer at uni-konstanz.de) wrote:


: Chris Boyd wrote:

: > Martin Chan (genetic_judge at hotmail.com) wrote:
: > : I have tried making a 6x gel loading buffer using the following receipt:
: >
: > : 0.25% bromophenol blue
: > : 0.25% xylene cyanol FF
(snip)
: > As a side issue, why not consider leaving out the bromophenol blue? It
: > will often obscure bands. Many gel photos are rendered cryptic by the
: > BPB hiding bands of interest. Solution: leave it out!  You don't need
: > it.

: Yes, Chris, you´re right that BPB often obscures bands in the range  between
: 300 and 600bp. I don´t agree, however, that the best solution is to leave it
: out completely. I perfectly get along with it, using a much lower
: concentration (around 0.025%, but to be honest, I just add a very little bit
: of the powder to the buffer). It is enough to be seen during the run, and
: doesn´t obscure bands. Plus, it gives you a good estimate of the progress of
: electrophoresis.
: If you really want to leave out the BPB completely, use Orange G at 0.05%
: instead. It runs ahead of all bands, so it doesn´t interefere with band
: interpretations.

Frank, thanks for the clarification. I should have been more explicit
in saying you can leave BPB out of recipes that include it AND a yellow
dye.  You do need SOME sort of tracking dye, if only to make sure your
gel is running in the right direction.  We, like you, use Orange G.
Using BPB at a much lower concentration as you do is a good idea: but I
have got used to living without it altogether. If you need to run a gel
a long way so that the Orange G runs off the end, you can always
include BPB in DNA-free lanes flanking the other lanes as a marker.

Best wishes,
-- 
Chris Boyd                      | from (but not \  MRC Human Genetics Unit
Christopher.Boyd at hgu.mrc.ac.uk  | on behalf of) /      Crewe Rd, Edinburgh
http://www.hgu.mrc.ac.uk/Users/Christopher.Boyd          EH4 2XU, SCOTLAND



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