Having problems with dimers and an answer from Paul Rutland for another question made me think that the fact that I store my PCR mix in the fridge might be the problem. I must add that in this mix I add both primers, so maybe dimers are being formed prior to the reaction during storage. Does this make sense?
Primer dimers usually happen at low temperature because the annealing regions are only a few bases. Some factors in storing mixes are primer quality (checked for low PD production) enzyme)....hot start enzymes can be stored with primers because they are inactive until after the start of the pcr but storing mixes with ordinary polymerases will often form a dimer. It can also depend on the freezer. Self defrosting freezers reverse the cooling process so the shelves of a defrosting fridge/freezer can get quite hot while it melds off the ice and this can make ordinary polymerases quite active
I was using an antibody covered hotstart enzyme. My mixes were being stored in the fridge though. Mix does not have a big amount of glycerol so I was afraid of loosing enzime with freeze/thawing. What do you think? And yes, primers not great. Unfortunately it is for diagnostic pcr and some organisms are just difficult
The storage duration of a PCR mix containing primers depends on several factors, such as the stability of the components, the storage conditions, and the intended application of the mix. However, using freshly prepared PCR mixes whenever possible is generally recommended to minimize the risk of degradation or contamination.
If you must store a PCR mix containing primers, you can typically store it at -20°C for short-term storage (several days to weeks) or at -80°C for long-term storage (months to years). However, repeated freeze-thaw cycles should be avoided as they can degrade the mix's components.
It's important to note that the effectiveness of stored PCR mixes can vary depending on the specific components and conditions, so it's advisable to perform validation experiments to ensure that the stored mix still yields reliable results before using it for critical experiments. Additionally, some specialized PCR applications may have specific requirements for PCR mix storage, so always refer to the manufacturer's instructions or relevant literature for guidance.
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