Smearing of PCR product

Nick Jacobsen jacobsen at cf.ac.uk
Thu Feb 13 15:02:15 EST 1997


Hi

You may want to try a number of things. Only add 0.5ng-1ng template DNA 
per reaction or do serial dilutions down to 1pg - this may cut down non 
specifics. Decrease your cycles to 30 or a bit less - to many cycles 
encourages rubbish to amplify. Also for a 260bp product you 
polymerisation stage need not be longer than 20-25seconds. 1minute at 
72C is encouraging all sorts of weird things to happen. Primer annealing 
can be reduced to 20seconds - this may discourage random priming. The 
above suggestions can be applied to both complex genomic 
targets(although you'll need more DNA) and simple targets. With simple 
targets you still get PCR artifaacts if you are to relaxed with your 
cycling parameters.

hope this helps

cheers

Nick. 




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