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Different Thermocyclers giving Different results - (Feb/25/2010 )

Hello,

I am having some confounding results regarding 2 different thermocyclers on PCR product formation.
Last year I was using a MJ Research PTC-200 thermocycler and using gradient PCR determined that the optimal Tm for my reactions was 50C. We just bought a new Aplitronyx 6 gradient thermocycler and I ran gradient PCR on the same reactions and found completely different results. The new optimal reaction runs at 57C and I am getting slightly better band intensity than I was on the PTC-200.

Is there such huge differences in Tm possible between thermocyclers? I reran PCR on both machines to test my findings and they are reproducible. I don't think anything is wrong with the machines downstairs since they are frequently used by 6+ different labs and noone has found anything strange.

I am also hesitant to say anything is wrong with the Amplitronyx 6 since it is a much nicer model and is brand new.

Any insight into this situation is appreciated

-Nebulus-

I wouldn't claim there is something wrong with the new machine (especially not if it gives you more product :) ). Different machines will heat and cool down the blocks at different speeds and in some cases, this can give different results in your PCR. Here in our lab, we have machines from different companies and when I know a PCR works on one machine, I always try to do it on the same machine.

-dpo-

Different machines will heat and cool down the blocks at different speeds and in some cases, this can give different results in your PCR


agree.. plus :

Is there such huge differences in Tm possible between thermocyclers?


yes, as we experienced different results with the same recation held on two different machines.
the reason was difference between Tm.
Attached File

-nightingale-

nightingale on Mar 3 2010, 05:50 PM said:

Is there such huge differences in Tm possible between thermocyclers?


yes, as we experienced different results with the same recation held on two different machines.
the reason was difference between Tm.

i would think that it is a calibration issue.

-mdfenko-

Regular calibration and check is mandatory as machines may shift in temperature and I also observed that temperature was not perfectly similar from well to well. Some brand with two heating blocks have one "pilot" controlled block and a "slave" block, it is then better to run sensitive experiment on pilot blocks.
Different machines might have different denaturing temperature (94C or 95C), usually pcr runs stay within limits for enzyme degradation. We experienced also troubles when transferring assays from one machine to another one of the same brand. This was mostly due to different temperature profiles: one machine was making (several degrees) overshots and undershots before reaching the set temperatures for denaturation and annealing, another one was counting time when still at 1C away from the setpoint, and it could take 20 seconds before reaching the set annealing temperature. increasing the annealing time and/or lowering annealing temperature by 1C solves the problem (-1C to -3C depending on reaction volumes). Similar troubles were observed when switching from gold/silver blocks to aluminium (less conductive). Another aspect is gradient machines: you don't only change the temperature with the gradient but also the ramping parameters. It is then critical to test one "broad" gradient and then a narrower one to get the actual optimum temperature.

-bucquoy-

This is normal. MJ Research PCR machines have two modes: block and Calculated Temperature Control. In the first one, there is a relatively great lag between sample temp. and block temp. In the second one, you enter the volume of pcr reaction, and machine overshoots heat thus making sure that sample reaches target temp. faster. if your other machine does not have such a function it is normal that you will see different results. even both machine use block mode, since their ramping time and block is different it will take different time to reach the same temp.

My suggestion is that you increase denaturation, annealing, and extension time to make sure that sample temp eventually reaches the desired temp.
Good luck.

-chromatin-