Dear colleagues,
one year ago we put our liquid primers in a high concentration into a -80 fridge. Can we still use them for the qPCR?
Primers are stable especially when lyophilised when they can be stored for many years. Liquid primers in water can suffer from acid depurination and possible exonuclease action if stored in water . Primers stored in TE stay alkaline and are protected from exonuclease contamination and degradation and can be stored for many years at -80. Used in pcr the primers are in vast excess and a small amount of freeze thaw degradation will not have any effect on the efficiency of the pcr reaction
The shelf life of an oligo is dependent on the temperature at which the oligo is stored, and how the oligo is resuspended. Temperature is the more important of the 2 variables. Generally, oligos should be stored at –20°C. At this temperature an oligo has a minimum shelf life of 2 years, whether it is stored dry/lyophilized, in TE buffer, or in (non-DEPC treated) water.
kindly see this file
https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/technical-report/stability-of-oligos.pdf?sfvrsn=c6483407_10
Anastasia Leonova
In my opinion, it's perfectly fine to use those primers. One should not expect any significant degradation of primers at -80 °C in a relatively short duration of time, i.e. one year.
Primers are stable especially when lyophilised when they can be stored for many years. Liquid primers in water can suffer from acid depurination and possible exonuclease action if stored in water . Primers stored in TE stay alkaline and are protected from exonuclease contamination and degradation and can be stored for many years at -80. Used in pcr the primers are in vast excess and a small amount of freeze thaw degradation will not have any effect on the efficiency of the pcr reaction
I agree with Paul's answer, and would add that the concentration of the primers is also a factor to consider.
Concentrated ones will be stable for years. If you use frequently, make small aliquots of concentrated primers, that you use.
If you need diluted primers, for example PCR sequencing etc., I would not make lower than 10pM, if possible use low binding micro-tubes; as the DNA will adhere to the test tube, and dilute with freeze/thaw cycles
Anastasia Leonova In my experience, primers stored at -20oC were still working after 10 yeas and more.
I agree with Paul Rutland
Yes, you can use it, as you mentioned your primers liquid stored at -80 one year ago, still, you can use it for a couple of years, I add to that, you can run qPCR and compare the result with your result that you got a year ago
Good luck
I suppose it will work well. DNA is conserved good for a long time, if it was not contaminated, and especially in deep freeze.To be sure you can try it on few samples.
In my experience for usual PCR not for qPCR, primers could be work well after 1 year in dH2O solution under -20℃ even without TE.
-20oC storage gives primers stability for about an year. You should not expect any significant degradation or quality loss in -80oC storage even after 2 years. The only concern should be the primers should be stored in multiple aliquots to prevent the whole stock to thaw & freeze repeatedly.
Best wishes
Primers are cheap. If you have any doubts about their reliability, toss them and order them again.
If the ones you kept in the -80 are the original stock, you should have no problem. However, if it is a dilution prepared by you, in water I have found that they lose a little specificity compared to those that have been recently prepared.
Yes, you can use. No problem. We use primers stored in -20 after year. No any problems occured. I feel that it will work well.
Primers protected from exonuclease contamination and degradation and stored at -80 can be used for many years.
- Badges
- Science method