Hi, I have some questions about qPCR mix preparation.
1) If I am pipeting a small volume (0,5 microliter) of my cDNA after adding master mix, I draw off the liquid to the wall of the PCR well, not into liquid, and then spin the plate, is that ok? If I tried to pipette into the liquid at the bottom, there was bubbles or/and droplets remaining in the tip again and again ... :(
2) Can I let my PCR plate get to be sterilised under an UV light in the flow box for 30 min? I want to sterilise my PCR plate before using because we have kind of weird stuff in the lab - a plate of which you can break off just desirable amout of rows. (By using your force or most often, non sterile scissors is used out of the flowbox. Remaining part is than given back to the plastic bag (!) for future reusing.
Thank you!
Lucie
1) Yes that should work. You can also put the cDNA first at the bottom, and then add the mastermix on top which will mix the samples without needing a spin.
2) UV sterilisation would kill bacteria/cells, but it will not necessarily get rid of contaminating DNA (only mutate it). You may want to spray DNA/RNA surface decontaminant on your scissors and manipulate the PCR plate under a DNAse-free cabinet.
I use the UV light from the hood to sterilise 96-well culture plates sometimes, but not for PCR purposes.
Hi,
Ok, if you add cDNA on the well and spin, it is fine. then do some pippeting and again spin to remove all bubbles. before spin tap with your finger to remove bubbles then centrifuge.
I agree with Martin, U can not remove RNase and DNase and just remove any alive contaminants. So I suggest you to use filter pippets and put all PCR tubes in a jar class and autoclave (if are resistance to autoclave) or use sterilized tubes.
Yaah.. I think no problem with that. But you should take much care during pippeting.
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