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Primers Freeze-Thaw - (Oct/10/2012 )

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Is it good not to freeze thaw the diluted primers (5 micro molars) when using it day to day?

Is it possible to store it in 4 degrees instead of -20 degrees?

-Mad researcher-

It depends a bit on what you are storing it in. TE or TLE is good for storing DNA at 4, but water leads to acid hydrolysis. PErsonally I freeze my working stocks and rarely have a problem, but then, I'm not doing a lot of PCR.

-bob1-

I dilute the primers in water. And i do a lot of PCR now.
I dont think its a good idea to store it at 4 degrees

-Mad researcher-

i always keep my working stock in -20C. so far so good. after 1-2 years the primers stil working great

-badguy-

I also store my working stocks of primers (10 micro molar) at -20C.

I've never had a issue with repeated freeze thaws for those primers I've used several times (I don't often use the same set of primers more than a few times, usually actually only once).

If I were you, I would make up my working stock of primers, and then store them in smaller aliquots so that the number of freeze thaws is limited. You could even store them in single use aliquots (say if you do 100 reactions a day, store just enough for this in each aliquot).

-leelee-

In a good buffer, double stranded DNA can be fairly stable at 4deg. That's not the case with single stranded DNA (primers) in H2O.

As mentioned above, freeze small aliquots at -20. Thaw at room temperature and then put primers on ice. Thawing on ice will damage primers more than at room temperature.

Freeze/thaw cycles can damage primers. When the ice thaws, the receding edge of the ice thaws and refreezes as it goes. Thawing on ice extends this process; extending the time it takes to thaw the ice increases the amount of time that the primers are thawing and refreezing. I don't have the reference but someone actually published a paper showing that thawing PCR reagents at room temperature is better than thawing them on ice.

-David C H-

I dilute primers in 10mM Tris pH 8 (actually a buffered water only, no chelators), stock and the working concentration too (nowadays usually working dilution is for about 50 reactions). When I know I won't use primers for a while I freeze them, when I'm using them continuously (like, one day, then two days later, on friday, then on monday and tuesday etc.) I don't freeze them, but keep them in a fridge. Since this continuous use can last as long as a month for example, the primers stay there for a month. Sometimes I forget them there even longer. They're fine. I think multiple freeze-thaw in that case would make them more harm.

The stock stays as is, I don't aliquot it, since I have many primers and having aliquotes for all of them would be annoying. Since this is pretty common and cheap reagent I usually don't care about them much, fridge primers work fine, but when the dilution itself is several years old (in that case with multiple freeze-thawing too, I don't keep primers in fridge for years) and I don't like the resuts, I make a fresh one. If the stock primers are themselves too old (like six years or so, depending on handling) and seem to be degraded even when fresh dilution is prepared, I just reorder.

-Trof-

We store primers at -20, and don't worry about freeze thaw. We keep DNA ladders at room temperature for many months when stored in TE, with no evidence of degradation. TE is your friend.

-phage434-

@phage434: Loading-ready DNA ladders in fridge are your friend ;) You can buy them (we finally did, since the price difference was dismissible I think) of you can read the composition of the loading-ready mixes and make your own.

-Trof-

Trof on Wed Oct 10 21:42:54 2012 said:


I dilute primers in 10mM Tris pH 8 (actually a buffered water only, no chelators), stock and the working concentration too (nowadays usually working dilution is for about 50 reactions). When I know I won't use primers for a while I freeze them, when I'm using them continuously (like, one day, then two days later, on friday, then on monday and tuesday etc.) I don't freeze them, but keep them in a fridge. Since this continuous use can last as long as a month for example, the primers stay there for a month. Sometimes I forget them there even longer. They're fine. I think multiple freeze-thaw in that case would make them more harm.

The stock stays as is, I don't aliquot it, since I have many primers and having aliquotes for all of them would be annoying. Since this is pretty common and cheap reagent I usually don't care about them much, fridge primers work fine, but when the dilution itself is several years old (in that case with multiple freeze-thawing too, I don't keep primers in fridge for years) and I don't like the resuts, I make a fresh one. If the stock primers are themselves too old (like six years or so, depending on handling) and seem to be degraded even when fresh dilution is prepared, I just reorder.


Do you recommend the primers to be kept in fridge when they are diluted using water?

-Mad researcher-
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