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Primers Freeze-Thaw

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16 replies to this topic

#1 Mad researcher

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Posted 10 October 2012 - 12:02 AM

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?
Cheers,

Mad Researcher

#2 bob1

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Posted 10 October 2012 - 12:08 AM

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.

#3 Mad researcher

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Posted 10 October 2012 - 12:48 AM

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
Cheers,

Mad Researcher

#4 badguy

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Posted 10 October 2012 - 01:27 AM

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

#5 leelee

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Posted 10 October 2012 - 01:28 AM

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).

#6 David C H

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Posted 10 October 2012 - 06:30 AM

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.

#7 Trof

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Posted 10 October 2012 - 01:42 PM

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.

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#8 phage434

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Posted 10 October 2012 - 02:08 PM

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.

#9 Trof

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Posted 11 October 2012 - 12:15 AM

@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.

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#10 Mad researcher

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Posted 11 October 2012 - 02:01 AM

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?
Cheers,

Mad Researcher

#11 phage434

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Posted 11 October 2012 - 03:51 AM

Loading ready ladders typically have bromcresol purple and xylene cyanole blue rather than orange-G as the loading dye. These blue dyes interfere with band visualization, and a far as I can tell are in every way inferior to orange-G. I've tried to convince NEB to switch, to no apparent effect. And why would I make an extra trip to the fridge if I can just keep the ladders on my bench?

#12 Trof

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Posted 11 October 2012 - 09:46 AM

@Mad researcher: In a water probably not, it doesn't keep pH. I was diluting everything in water while working on diploma thesis, until the moment some primers just stopped to work even though they were freezed all the time. I took advice about the Tris and dilute everything in it ever since.

@phage434: Yes they do, but our ready load mix from NEB contains only bromphenol blue and I didn't ever notice interference with ladder bands. I made my own before that, you can adjust the amount (or type) of dye.
I had more problems with the bromphenol blue loading for samples, there are many those that run around 400 bp range of bromphenol, but I adjusted the concentration and made a special "visual aid only" loading with just a bit o xylene cyanol for samples that are extremely faint in that range. Orange-G runs far too quick for our use, so it's unusable for longer runs. And what also not unimportant, we don't have it ;)

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#13 Ameya P

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Posted 11 October 2012 - 10:10 PM

I would disagree with all our experts here.

Working primers diluted in water and stored at 4oC work perfectly fine for up to 2 years (that's how I long been working in this lab). Actually, even my stock primers are diluted in water but kept at -20oC

Some working primers I use on more or less a daily basis but other dont come out for months together.


and about the ladder, we keep that too on the bench, with no issues. I have gone ahead and diluted ready-made ladders with equal amounts of water and they work fine for me. So, lot of money saving and also effort saving :P

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#14 Mad researcher

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Posted 11 October 2012 - 11:49 PM

@Trof - Could you give me the recipe for 10mM tris ?
Cheers,

Mad Researcher

#15 Trof

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Posted 12 October 2012 - 06:16 AM

I would disagree with all our experts here.


Well that's not actually a diagreement, that's just a different experience. I had mine working for years too, then once just degraded.
Actually with the primers it's bit like with the computer disk backup, everything is so long so fine, until one day.. it's not.
It's everyones decision if he wants to risk that, but same as with backups, once you got your primes degraded/data lost, you kind of change a view on these things.

@Mad researcher:
100x diluting the 1M Tris pH8 stock with milliQ water (100 ul for 10 ml of buffer), I aliquot it into 1.5 ml tubes.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






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