Does it affect the efficacy of qPCR data if I prepared syber green with primers and kept it for 1-2 hours before adding cDNA and running QPCR?
Thanks
Hello Dr. DG,
Since SYBR green binds preferentially to double stranded DNA, creating a master mix or mixing with primers prior to qPCR is not a problem. The binding to RNA is low and might have s little extra background, but be sure to run a control well with the SYBR green and primers to see if the background has increased and the. If it does increase the threshold limit or subtract the background fluorescence. You could also compare "fresh" and 2 hour old mixed PCR mixes to see if it made a difference if you want to convince yourself. Some kits are actually sold premade for pathogen detection with primers and dyes commercially.
Hello,
The critical component to your qPCR is your polymerase, if it's 'hot-start' (it should say this in the product description pamphlet) then you are good, because it only gets activated at the warm-up step in the machine. Bear in mind that some hot-start enzymes might require an extra 3-5min incubation at 50C or so before starting the regular protocol at 92C (again it should state this on the product description). In my experience 1-2 hours is nothing, as long as you have kept the plate on ice while waiting!
Good luck
As any fluorophore, SYBRGreen is subject to photobleaching, so you should minimize light exposure if you have a waiting time before the run.
In regards to setting up the reaction beforehand, 1-2 hours are perfectly fine if you use a HotStart polymerase as Mae points out. I've even got good results on fully set-up reactions after overnight storage in the fridge (someone stole my booked spot at the qPCR machine and I really did not want to wait around another 1.5 h to put it in as it was already late).
I second Petra. Having a hotstart polymerase is good, I even strored reactions over the weekend at 4°C and did not see any difference. I would bet that this would even be the case when the reactions are stored at RT.
Light exposure in the lab is not a big problem for SYBR Green. No need to keep it particularily dark. I just would not keep it upon the windowsill in direct sunlight ;)
It is very useful information I got from all of you guys,Thanks a lot.
Thomas Weppelmann: the idea of having control well is very good.
So u commented on the stability of SYBER GREEN, what about the primers, r they safe, de-polymerization of the primers might be a matter of concern?
Primers are added at a vast overshoot, the same applies to dNTPs. Even if there was some degradation, it will be negligible.
hi dear
for me iam using template DNA for qpcr and i prefer preparing the master mix freshly ; it is only 20 minutes before using pcr
SYBRGreen is light sensitive, exposure to light should be minimize in case you still have some time before running.
However, for 1-2 hrs keeping period is very Ok. We have got good results on the whole set-up reactions following overnight storage in the 4 degree with a proper covering.
Good luck
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