Hello,
I'm performing a qPCR experiment in 10 μL total reaction volume. I wonder if it's obligatory for the total reaction volume (including the Supermix, primers, template DNA and water) to be precisely 10 μL.
Can I skip the (nuclease free) water adjustment step or would that impair the reaction performance?
I'm asking that because it seems that I've contaminated the water I'm using with some DNA of my samples so I thought if I skip that step I might decrease the chances of contaminating my samples. I'm still a lab dummy.
Thank you very much in advance!
if you think that you have pcr contamination because the blank control sample amplifies the right size product then you need to sort out which reagent is contaminated before you run any more reactions. PCR contamination is a curse and any reagent can be contaminated. What makes you think that it is the water that is contaminated?
Thank you for your answer. And yes, it is exactly the blank control that shows amplification at the expected size.
I think it's most probably the water which was contaminated because the supermix I used was a brand new one as well as the primers (I diluted them at the needed concentration right before I ran the experiment). But you're right about that the other reagents could have been contaminated.
I guess that I'll just redo the experiment without adding water and see if it changes anything but do you think that it would affect in any way the reaction efficiency (maybe the reagents would be too concentrated without water addition?).
You need to be extremely careful, accurate and precise with your measurements for qPCR. You can NOT have variations in sample volume or your data are meaningless.
The water is an important reagent as the final concentrations of the PCR mix depend adding water to bring it to an exact volume. The only way to not add water is to dilute out your primers or DNA so that you don't need to add any water to get to the desired 10 microliters.
You should talk to your advisor/mentor. You shouldn't be starting with qPCR as a first project since it sounds like you need to learn about how to do regular PCR.
yes I do think that the volume is very important..one aspect is magnesium concentration which will have a large effect on pcr . Too high a concentration allows non specific amplification. Can you borrow water and pipettes from a worker distant from your working area to set up a new amplification and ideally their mastermix as well and do a run with a good number of blank samples to try to isolate the cause of the contamination before having a major clean up of working areas, pipettes, reagents anywhere where gels have been run or tubes opened which contain pcr product
Thank you, Katie and Paul, you answered my question. I will be more careful and will definitely not try to skip adding water to my samples in the future.
I forgot to mention that I also had a blank control sample without any water in it (only mastermix & primers) which did NOT show any PCR product so maybe it really was the water after all.
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