I have always found primers to be very stable at 4c especially when dissolved in TE ( not water which allows depurination). The evidence is ( file attached) that they are even stable to less than 30 cycles of freeze/thaw but I would normally avoid freeze thaw cycles so if your fridge has automatic defrost I would avoid storing close to the cooling coils . 5% glycerol as an antifreeze should be fine and not interfere with primer binding in the reaction mix and a small amount of albumin may help to block non specific binding to the plastic of the tubes. After prolonged storage of small volumes I would expect evaporation and re condensation of water at the top of the storage tube so before using the aliquot spin the storage tube for a few seconds at any speed to ensure that the total volume is now at the bottom of the tube I attach a file on oligo handling from the IDT website for your interest which suggest that 6 months storage in TE should be fine.

Hello Mischa

To my knowledge and based on references, primer mixes are stable for approximately 1 year in 4c either in water or TE buffer.

Make sure that the pH of TE is 8 because ssDNAs are sensitive to pH.

You can make working primer mix by diluting the stocks in TE again.

Primers resuspended in water are less stable compare to dissolve in TE buffer.

The impact from the type of storage medium used is marginal at -20°C, but becomes more important at higher temperatures. However, primers resuspended in either water or TE buffer are stable for at least 60 weeks at 4°C or 30 freeze/thaw cycles at -20°C. I attached IDT Oligonucleotide Stability Study.

I got a gratefull experience from previous answers.

in our lab we reconstiute the primers in nuclease free water and store in -200C and it works well for at least one year. I do not prefere to store primers mix for multiplex PCR because if the mix does not work. I cannot determined which one is not working. The primers should be sotred seperataly. Multiplex PCR of Pasteurella multocida did not work well when we used the extracted genomic DNA eluted in TE

Thank you Ashraf Abbas , Iryna Goraichuk , Seyed Elyas Meshkani and @Paul_Rutland for your valuable input. For standard oligos we do not have a problem (some are 5 years old and still work nicely), but i have degenerative and labelled oligos in mind in particular. The degenerative oligos start to cause non-specific amplification after +-25 freeze-thaws and the labelled oligos range between 25-35 freeze-thaw cycles. Thank you again for you valuable input will have a look at the articles you have all provided.

Dear Mischa

In case of labeled oligos, they should be resuspended in TE buffer.

Make sure to store them in dark tubes or if you are using transparent tubes wrap them in foil.

UV light (from PCR cab) more than 20 minutes may create ozone which poses a negative effect on the shelf life of the labeled oligos.

Elyas is right about labelled oligos. Degenerate oligos should not be a problem as each oligo only has one sequence. When you order a degenerate oligo you get 2 oligos if the degeneracy is 2 bases or 4 oligos with 2 degeberate positions etc but all of the primers are full length and single sequence you just have a mixture of full length oligos. The IDT information tests freeze thaw to 3 cycles but I think that the safe option is storing at 4c for use and just the stock frozen long term and not thawed very often

Hi Mischa,

The primer storage for single primer pair and multiplex primers is two different issues.

For the past eight years I have been following these points as SOP for primer dilution and storage. So far, I have worked with three different multiplex PCR, most of them hold 3-7 different pairs in single tube. The primary challenge in multiplex primer storage is consistency in amplification.

It is always ideal to store the Forward and Reverse primer at (10 picomole conc) in two separate tubes. Majority of the time, we used to dilute with TE buffer, pH8.0. Upto my knowledge, primers diluted with TE buffer can be stored for atleast twenty years (in -80C) without multiple freeze-thaw cycles. The major stock (100 picomole) usually stored (in aliquots) at -80 C and vial taken for dilution (minor stock?) at -20C. Primers at (10 picomole) usually stored (in aliquots) at-80 or-20C and 4 degree.

You should remember one important point: more number of primer means higher chance for primer-primer binding especially at lower temperature. Hence, it is not advisable to store multiplex primer pairs in single tube for longer time in larger volume which increase the probability of primer dimers and frequent freeze-thaw effect. Never store multiplex primers (either as separate tube for each primer or primer mix) greater than twenty microliter. Further, the selected volume to store at 4 degree Celsius must be within three or five time freeze-thaw cycle. If you follow this procedure you don't require any additive in your primer mix. If primer sequence itself disclose the possibility of primer dimers, it is better to store that specific primer pair or primer as separate tube. If there is no or least chance for primer-dimer (based on primer seqeunce) you can store the primer mix with 3-5% glycerol. However, you have to optimize the PCR with the appropriate concentration of glycerol.

I hope these suggestions may help you in some form.

I agree with Paul Rutland and and disagree with Narayanan Kalyanaraman . Any primer-primer interaction should be broken down at the first denaturation step. Furthermore, the kinetics of primer-primer interaction should be at least as rapid as annealing to template, storage should make no difference, and if primer-primer interaction is a problem, a hot start protocol (and, preferably, redesign of primers) would seem mandatory. I would argue that making and storing primer mixes should lead to greater reproducibility, particularly in the case of multiplex reactions.

That said, I haven't tried. If the voice of experience says the reverse, then the voice of experience is right.

Dear Andrew Jenkins,

You are right. I thank, Andrew to point out my mistake at the earliest. It is my fault to communicate it properly. I addressed the problem, but didn't addressed the existing solution. The primer-primer interaction can be easily broken down at initial denaturation or it can be easily overcome by using Hot start enzymes.

The final message is storage of multiplex primer mixes is advisable at lower volumes (less than twenty microlitre), at lower concentration (at ten picomoles) and with glycerol 3-5%. People like me, working with multiplex PCR (for atleast five year) usually store the main stock (at -80C) in individual tubes not as multiplex. Whenever required or working stock is in shortage, we prepare multiplex primer mixes at larger volumes (say 100uL) and store in aliquots (at 20 uL) which depends on no of experiments or requirement. The storage of multiplex primer mix (seven pairs) for longer time at four degree Celsius with more than five thaws will produce inconsistent result. I speculate, the stability of single primer in TE buffer and multiple primer mix in TE greatly differs based on the method of primer purification. The level of impurities remains in the primer while synthesis determine the rate of stability (as a multiplex).

Thank you Andrew Jenkins , Paul Rutland , Narayanan Kalyanaraman . Valuable inputs. As mentioned previously, these are for high throughout processing of various services and it would be easier to store solutions at 4degC. I am working on ways to optimise our workflows in an attempt to maximise productivity, reduce hands on time, reduce the need to walk into our walk-in freezers as it causes fluctuations in temperature that may affect the integrity of our stocks, reduce costs and if we could store the oligos (simplex, degenerative and multiplex) at 4degC it would improve productivity a great deal. While this is not currently a problem, I would like to start testing various stabilisers to mitigate risk. I have discussed this with a number of chemists, molecular biologists and commercial laboratories and the input has been mainly on the presence of detergents to minimise the adherence of the oligos to the plastic consumables and optimising the concentration of TE to prevent depurination. I think we will start there.