Hi everyone,
We were working for years with GeneAmp® PCR System 9700, emulating the 9600 ramp. And now we have changed to a Veriti Thermal cycler, emulating the 9600 ramp as well and using the same program as we have always done. The fact is that some routine analysis that worked well with the first thermal cycler started to fail with the Veriti, and the program that lasted 12 hours, with the Veriti lasts 10 (2h difference). We suspect the problem is that the ramp time is different between thermos, as the program is exactly the same.
Can anyone give us some tips to optimize this new thermo? It's normal this kind of diference?
Thanks!
hi, dear Laia Closa Gil
This was my problem, I used two model Thermalcycler for a routine study. and finally come to this conclusion that, I have to re-setup my program in the new PCR.
I think that you are probably correct that the ramp conditions are different and some of your reactions may have relied on overshoot and undershoot of rapid ramping so that , for instance , when the sample moves from 72 to 94 the overshoot may momentarily take the reagents to 95 before settling at 94 and at annealing if set at 58c the fall from 94 to 58 may drop to 56 then come back to 58. So assuming there has been no change in reagent volume and type of tube/plate used I would ,for the failing reactions, drop the annealing temperature 2c and raise the denaturation temperature by1C and keep all of the dwell times the same. If this fails you may have to run gradient pcrs again. I assume that you have checked the ramp rates of the 2 machines and set the ramp rate of the Veriti to match the 9700 but the 9600 and 9700s that you used previously may have been getting old and the temperature profile as set may not have been matched in reality by old circuitry and peltier blocks hence my suggestion to make the profiles a bit more conservative. Check also that the default volume for the Veriti is the same volume as the 9700 as some old machines had a default volume to calibrate temperature profiles of 50ul but the more modern machines may be set for 25ul liquid volumes as default. Changes in volume will change the rate of temperature change and even on rapid cycles the ability to reach the desired temperature in time
Dear Paul Rutland thanks for your answer, Ive checked the temperature during the ramps and you were right about the overshoot and undershoot because of the rapid ramping. We will decrease the ramp velocity in this new thermo to avoid it. I hope this helps!
Dear Laia
If you want to reduce the over and under shoot .. you have to set in the Veriti™ 96-Well Thermal Cycler the sample volume to the minimum as possible because more sample volume to heating and cooling means normally more over and undershoot.
best
Stefan
Dear Stefan, our sample volume for this routinary PCR is 10 µl, do you think we need to decrease it even more? I realised that decreasing a bit the velocity of the ramp I can reduce the over and under shoot to 0,1º (94,1º or 61,9º), maybe it's not necessary to change the volume then. We will perform an experiment to check both things anyway.
Thank you!
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