Help with Real Time PCR Well To Well Variation
Posted 15 March 2009 - 04:44 PM
Posted 15 March 2009 - 05:38 PM
Posted 15 March 2009 - 06:07 PM
Posted 16 March 2009 - 07:55 AM
Posted 17 March 2009 - 05:37 PM
Assuming that your pipettes are calibrated, the instrument is clean (when you take a look at the block using the instrument's optical system you do not see any bright spots), and you have a good assay, I am going to guess that maybe you are not pipetting your solutions well? I always reverse pipette the qPCR master mix since even at 1x it is viscous. Also, none of my volumes is less than 2 microliters. Since you are doing technical replicates hopefully you are preparing the whole reaction and then pipetting the whole thing into each well. If you are adding the master mix first and then the template to each well you may be having issues with pipetting small volumes.
My painful experiences in real-time PCR totally and absolutely support ivanbio's points. Try to "preparing the whole reaction and then pipetting the whole thing into each well", this is very important to eliminate well variation. And other suggestions are:
1. Whenever you aliquoted any solution, make sure to mix them beforehand.
2. When you pipette your solution, make your pipetting same each time. I mean if you pipette all solution in tips out for the fisrt well, then the same for the rest two and if you leave a little solution (around 0.5-1.5 microliters for 100 microliters tips, caused by the pipette) for the first, then the same for the rest two.
In my lab, we prepare qPCR mix by ourself not commerical qPCR master mix. As long as we keep above points in mind, we could eliminate well to well variation. May it help you and good luck.
Posted 18 March 2009 - 10:51 AM
Currently, I have well variations on a plate within ~0.1 Ct when pipetting with single channel pipette (with 8channel its not that accurate but also ok.) and I'm adding master mix and cDNA separately. Maybe also your reverse transcription reagents "disturb" your reactions?
Well variations of more than 1 Ct are unlikely caused by imprecise pipetting. this assumes you put in one well > double amount of template compared to the other well.
Posted 20 March 2009 - 08:39 PM
Posted 23 March 2009 - 07:37 AM
Posted 16 May 2009 - 08:37 PM
Posted 18 August 2010 - 03:08 PM
Posted 18 August 2010 - 04:59 PM
not sure if people are still paying attention to this thread: but any further thoughts on the necessity of spinning down plates before putting in the thermocycler. A post-doc in my lab has reasoned that it doesn't really matter with her hotstart assay because the heat will disrupt the bubbles and cause the solution to evaporate and then condense regardless. Is this faulty reasoning?
She has a point, but it's good practice nonetheless.
e: It's not like we can ever see it while it's happening sadly.
Edited by Whiskeybent, 18 August 2010 - 05:00 PM.