PCR and loading solution

[Mary Clare Tracey]

MELANSOND wrote:
> 
> i find it hard to believe that one can add the dye and sucrose to the PCR
> reaction mixture without severely inhibiting the reaction, however, there are
> many things I don't know. So if this truly is possible I would like to hear
> about it as well.  In the meantime...  I would suspect that one should add the
> loading dye to the PCR reaction tube AFTER the reaction has taken place,
> immediately before loading the gel.  The concentrations depend on the
> concentration of the stock loading dye (10 X, 6X etc), but one would ideally
> like to add 1unit of dye / 4 units of reaction volume, to achieve a final
> concentration ratio of 1:5.

I posted last week but I'm not sure if the post made it to the wider 
ng. I use the dye Cresol Red in my PCR reactions. Stock Cresol Red 
(CR) is made up at 10mg/ml. I add 25 microlitres CR to a final 
mastermix volume of 750 microlitres. I also use glycerol in the PCR 
reaction, 125 microlitres per 750 microlitres final volume. I use this 
mastermix diluted 1:1 with the template DNA. I have never had any 
inhibition of the reaction. I am using 10 microlitre PCR reactions and 
load the entire reaction onto the gel. This method is routinely used 
in many HLA Typing laboratories around the world and is very 
effective.

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Mary Clare Tracey		email: maryclareatkokako.wnmeds.ac.nz
Wellington     **NOTE: change the at to @ for reply. Avoiding spam!** 
New Zealand