Sproadic PCR failures - (Mar/05/2008 )
So it goes like this...
After optimizing my PCR components and conditions I set up 96x10ul reactions to assess the amplification yield of a new thermocycler (for quality assurance we do this every month just to verify that the cycler is working nicely). Of the 96 reactions that are set up, approximately 15% fail in a random manner that is not restricted to specific locations within the thermocycler block (which by the way is gradient and therby controled by three different peltiers). The 96 reactions are set up from a big master mix employing the same DNA of known (good) quality and same reagents. The remaining reactions work very nicely with sharply defined, bright bands running in the expected place (1.4 kb).
Does anybody have any clue as to what might be interfering with these reactions? I've been surfing the web in search of answers and have come up with problems relating to glove powders, fractured pcr tubes, DNA globs of non-homogenized samples and childish demigods...can anyone confirm this or suggest otherwise...
God I hate it when I get stuck with these problems... Please help!
Do you spin the plates down to make sure the mix is at the bottom of the wells?
In a similar vein, is your hot lid working? Try using mineral oil on top to prevent evaporation to see if the hot lid is an issue. Are you sure the master mix is well mixed?
maybe some dust has entered some of the wells of the plate.
there could be variation in the amount of PCR mix added to each well. It does happen with my multichannel if I don't tap the tips down.
Might the sticky film on the PCR plate be failing to form a perfect seal around every well... but you can see this.as well that have not been sealed properly will lost volume after the PCR reaction.
And, yes is the master mix real well mixed. THere could be variation in within the tube.
Master mix is vortexed for 15 secs before aliquoting to 96 tubes...I'm pretty sure its ok. Hetaed lid on the cycler is working OK. No variations of dispensed volumes (been doing this for 7 years now)...It seems last batch of gloves came with powder and no one told me about it...giving PCR another try with powder less gloves...will let you lot know. I really appreciate your input...Best.