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I need to break this cycle of PCR issues - (Oct/25/2012 )

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Ah, I see. I've heard of pre-mixed master mixes but have never used one myself - I think my project leader would not be too happy with me asking for commercial mastermix when we have oodles of PCR reagents in the I suppose I might have to just troubleshoot until i can get my hands on the pre-made stuff ;)


If you can't use a commercial master mix, I'd suggest making large batches of your master mix. Then the variation will be in your sample, the program, the tubes, or the cycler. Where does your water come from?


Previous users gave good tips, are you adhering to all the 'rules' for a good PCR? e.g don't freeze-thaw aliquots of primers, dNTPs, DNA template etc. multiple times? Also, using different primer concentration aliquots (for ex. I aliquot 10micromolar, 20 and 50, and sometimes the 10's will go off) can sometimes affect the reaction. If you store in TE, that can affect downstream efficiency- do you always add the same amount of starting template to your reactions, or do you 'dilute' out? The presence of inhibitors from that source may contribute if so.


Hmm, rules.. I use mastermix that is in the fridge all the time, I only aliquot DNA if it's in large quantities and used very often, only have one stock concentration of primers and one "working" (the working one usualy in a fridge if being used regulary, if it stops working I make new ones, if it still doesn't, I order new primers, happend once, the primers have just had seventh birthday), I store everything in 10mM tris pH 8 only and if the DNA concentration is within certain range I always add the same amount of template,1 ul.
So rules.. well.. ;)
(this of course apply to classic PCR only because I basicaly only care if it amplifies fine or not, and in vast majority it does)

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