I wanted to run qPCR, I prepared all samples on plate (everything is mixed there - Brilliant III Ultra-Fast SYBR mix, primers, cDNA) and thermocycler broke down. I don't want to waste a week of my work, but I want my experiment to be accurate. All components are stored in -20C when separated. Should I store my plate in -20 or in 4C and what is the maximal safe time for such storage?
Hi Aleksandra,
Store it in 4C in dark place (wrapped with foil for example) for maximum of 4 hours.
Hi,
You can store your ready-to-amplify samples in 4oC in dark place as Rana said, but from my experience you can prolong the storage time till over night. Everything should be ok.
Since you are dealing with Taq and not an RT enzyme, and cDNA (as opposed to RNA) - your plate can theoretically be safe to run the plate for a week or more if stored at 4C in the dark in the meantime. cDNA and Taq are very stable - especially if you have taken care to not introduce nuclease and/or protease contaminations during plate prep. You will find the reaction components in your case a bit tougher than most say they are.
Too late. I have it in -20 ha ha ha ha ahha. I will run it tomorrow (I will add a bit of mix) anyway and let you know. I know they are tough, because I prepare all samples without ice and people are crazy while dealing with cDNA, they run with buckets of ice. Everything works very good for months without any problems and is much cleaner on the bench ;)
As long as the reactions did not freeze too hard at -20 (e.g. glycerol content of the final reactions probably partially helps in that regard)... but, cDNA and Taq are tough against freeze-thaw as well (tougher than many investigators think). You will be fine. (Let us know what happens for sure).
Hi. It worked fine. Now I am tempted to prepare many plates and freeze them down. I kept my plate in -20C for 2 days.
When you say it worked fine, are the CT values also comparable to previous runs? And did you let the plate thaw before running it in the machine?
Excellent - cDNA and Taq (and other reaction components) are indeed probably tougher than most of us think. I have also experienced good results with delays in plate runs, although I have not stored them at -20C, I have stored at 4C for up to 4 days and all Cq results were perfect: standard curves, inter-plate calibrators and samples. Using a good mastermix (with glycerol as an included component; which is often the case anyway) is probably a major factor - as is good clean, careful sample preparation and nuclease-free technique(s). Great hearing that it worked Aleksandra ~ food for thought.
Lucas brings up a good point...Were your CT values comparable to previous runs? If so this could turn into an option in case we have "back up" or a line on people wanting to use the thermocycler. Looking forward to hearing back.
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