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Pre-Preparation of qPCR plates - Pros and Cons (Aug/02/2006 )

Hello People,
I am at the end of my PhD program and beginning my thesis, which means I dont have a lot of time to do intensive benchwork. BUt I still need data using RTPCR. SO I deviced a method where I come in the weekend and prepare cDNA on a 96-well plate. Then add my primer mixture and cDNA in to a new plate and freeze it at -20 (Cant do -80 bc of space issues). Later, I take the plate out, thaw (usually takes less that 5 min bc of volume) and add SYBR green Master Mix using a multi dispenser pipette, and run the plate. I have done 2 plates so far. One of them worked very well. In the other none of the samples amplified (RNA might be the problem!).
From my limited knowledge of the RTPCR theory, I do not see a contraindication to what I am doing, but I thought I will ask the council before I went ahead and started doing it on a regular basis.

WHat say you?

-Brown girl in the ring-

first of all, I'm not really understanding why this is faster?
secondly, I think in qPCR as in most of science, the more you manipulate your samples the less likely you are to get good results...seems like an extra freeze-thaw of your samples at least

so where are you saving time?


I have about 60 tissue samples. I did all the cDNA in one go in a 96 well plate. I basically didnt want to freeze and thaw the cDNA everytime, so I aliquoted this along with the primer in various 96 well paltes that I run RTPCR in. Then I can thaw only the plate that I am going to run. I add the SYBR green master mix right before I put the plate in since the SYBR has to be stored at 4C. That way it prevents repeated freeze thaw of cDNA.
Is this OK?

-Brown girl in the ring-

oh, I see


I don't see any problem with that. it sounds like a good idea, actually

I don't know why you were having problems with some of the plates tested that way, though?



We used to set up 4 or 5 qPCR plates at a go with all the reagents and then place them in the -20. Take out a plate from the -20 when the last run finishes and let it thaw, spin it down and put on machine. It is handier this way if you have a thesis to write or need to head home in the evening as it means you only have to leave the computer/house for five minutes to change plates in the machine after making all the plates.