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PCR and loading solution

Mary Clare Tracey seebelow at for.my.address
Sun Oct 20 19:36:18 EST 1996

> 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 

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

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