Does anyone know if its possible to prepare the master mix and add all in a real time pcr plate, put the plate in freezer or refrigerator and then run the reaction the day after? Does it affect the results?
As long as you don't add polymerase you should be ok. However, keep in mind that the polymerase only acts as a catalyst for a reaction that could occur spontaneously. Most molecules in suspension tend to interact with each other (hydrogen bonds, Vander Waals forces, etc). Your primers might anneal to available templates, your dNTPS might anneal separately just by chance and the overall efficiency/ specificity of your PCR might decrease.
As long as you don't add polymerase you should be ok. However, keep in mind that the polymerase only acts as a catalyst for a reaction that could occur spontaneously. Most molecules in suspension tend to interact with each other (hydrogen bonds, Vander Waals forces, etc). Your primers might anneal to available templates, your dNTPS might anneal separately just by chance and the overall efficiency/ specificity of your PCR might decrease.
I agree with Maria. I think if you meet a problem just after mix everything even RNA or DNA, it's also possible to store your preparation about one to 3 hours in the freezer until your resolved your problem like disruption of machine or courant failed.
But if you want to go quickly because you run a lot of RT-PCR, you could pepare mix with all ingredients without DNA polymerase and conserve at -80°C. When you are ready, you defreeze the mix just add the DNA pol and dispatch in the well of plate and RNA.
Depending on what reagent you are using (hot start taq with antibody/aptamer and other preservatives), you may even be able to keep the prepared reaction at room temp. I've been using BioRad's Sso advanced SYBR green mix and they claim that you can keep a prepared QPCR plate at room temp for days (they have a 384 well qpcr machine with a plate loader).
http://www.bio-rad.com/prd/en/US/adirect/biorad?ts=1&cmd=BRCatgProductDetail&vertical=LSR&catID=M87F92IVK
Not out of choice, but I did use master mix after being stored at -20 for couple weeks. I compared to freshly prepared master mix, and the results were comparable. For experimental consistency, I wouldn't do that on a routine basis. But it was nice to know..if a machine fails on you..or you prepared more than you needed, it is ok to use it in a future assay.
From my experience with master mix sybr green, the results are a little bit different.. I do not reccommend as a time-safe solution.. It happen to me as someone mentioned, concerning a problem with the equipment, and the plate was in the freezer during 2h. I ran that plate and another one similar, and results were different.
I agree with Issaka Maman. From my experience, prepare mix with all ingredients without DNA polymerase and keep it first before you ready to run the experiment. For me, after the preparation, if the equipment not ready, I will keep the plate in the freezer (4 oC). The results were same even I run for the second time (re-run the same plate) and prepare a new plate with same DNA polymerase. However, the intensity was reduced when re-run the plate,
I use Qiagen's QuantiTect SYBR Green qPCR master mix. The polymerase needs to be activated 15 min at 94°C before the actual qPCR. I routinely either put the plate directly into the machine, leave the plate 2 - 4h at 4°C or even store the plate over night at 4°C. I haven't compared results side by side though. Still, I haven't seen any alarming differences between those conditions.
Had to do this a couple of times. I highly discourage freezing. 4 degrees is more than enough for a 12 hour wait. However, I worked with highly abundant products, and this might not apply if your sample is diluted and too little transcript. Good luck
I prepared my plates with cDNA, master mix, etc and ran a couple straight away, and froze the rest. I found amplification was very inefficient for the ones I froze.
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