I tried to scale down the component in reaction (from 20 µL to 10 µL using Bio-Rad Universal SYBR Green) for saving material. I wonder if I can get precise results or not with a down-scaled reaction because the RealTime PCR machine in my lab was calibrated for using 20 µL of PCR reaction.
Thanks a lot for any advice/suggestions.
P/S. For testing, I prepared 45 µL of master-mix, then separated it into 3 tubes to make 20 µL, 13 µL, and 10 µL of reaction. The result shows a little bit different in Ct and quite big different in RFU.
Yes it is. It is not so wildly different that it will have a large effect on your samples (which I presume are the same volume anyway, normalising for any residual effect)
So that will be completely fine
If you are measuring relative expression between different conditions/samples (deltadelta CT method), then no problem !
Just make sure your melting curves look fine.
Yes, as members also said, you can go for a 10ul reaction, we have done many times, even initially we tried the same sample with both volumes, got the same result no difference. Primer concentration, Tm, cDNA concentration should be good from your end. The rest all will go fine.
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