Hi, my question is about the heating of thermal cycler machines and I hope some of you had experienced a similar thing previously. There are two thermal cycler machines in the lab(BioRad) and for some time I have been thinking that their heating patterns may be unstable. Last week, I conducted a PCR with the same enzyme(Phusion master mix) and the same DNA in 4 different tubes. I put 4 tubes(together with negative controls) into the same thermal cycler fraction, 2 of them worked and others did not. Then, I prepared exactly the same reaction mixture and this time put the tubes into another fraction, this time all worked perfectly. I was skeptical about it but now I am kind of sure. Why do you think that this happens? Do these machines need controls periodically? Bu the way, some in the lab suggested this may be due to the lids, one lid is kind of broken but still closed. I try not to squeeze too much because PCR tubes can be crushed during the reaction(I experienced before). Do you have any suggestions and/or comments? Thanks in advance.
Most pcr machines these days use Peltier junctions for both heating and cooling but some are not great at cooling so sometimes,especially if the machine is routinely used for 4c holds overnight after pcr a junction can fail. Junctions control blocks (rows/columns) of the heating block so you can get a situation where 1/4 or 1/3 of a block is failing or has failed.. You could try a pcr that usually works in one row all the way across the heating block and see if you get a result which looks anything like ++++++---+++ where + is pcr working and - is no product/fail across the block
Most pcr machines these days use Peltier junctions for both heating and cooling but some are not great at cooling so sometimes,especially if the machine is routinely used for 4c holds overnight after pcr a junction can fail. Junctions control blocks (rows/columns) of the heating block so you can get a situation where 1/4 or 1/3 of a block is failing or has failed.. You could try a pcr that usually works in one row all the way across the heating block and see if you get a result which looks anything like ++++++---+++ where + is pcr working and - is no product/fail across the block
I completely agree with @Paul about failure of peltier junctions due to long lasting 4 Degree incubation after the cycle.
Other thing I would like to indicate that low quality tubes ( used to set reaction) may leave a bit of plastic due to excessive heat which accumulate in well. And when you keep your reaction tube in the same well your tu e doesn't get proper heat causing failure of reaction.
Just check each well of your heating block with torch, if anything is wrong there.
You may get the instrument calibrated from manufacturer if peltier junction failure is the reason.
I agree with Paul Rutland's suggestion. I may add that introducing a 4 °C hold overnight a a final step is useless and will decrease the lifetime of Peltier elements needlessly, in part due to condensation. A 15 °C hold is quite all right to keep amplified DNA intact overnight.
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