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Primer Reconstitution--does temperature matter?


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5 replies to this topic

#1 Tatooinedweller

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Posted 16 July 2010 - 10:05 AM

Hello,

Two months ago I reconstituted two sets of unlabeled primers from Applied Biosystems and made aliquots. One set of primers worked, the other I wound up not using. However, now a colleague has found one of the aliquots not to work (and is assuming they all don't--she's a post-doc and I'm the student, so I'll trust her on this one). They haven't been used before, so the obvious answer is that I screwed up the reconstitution somehow. I'm certain that I added the correct water and the correct amount, and doubly certain because the other set of primers (which I did simultaneously) did work. My adviser thinks that the problem is that I reconstituted with room-temperature water instead of ice-cold water, and some minor composition difference in this set of primers makes them more vulnerable than the set that worked. This doesn't seem a very satisfying answer to me, especially since I can't find a protocol that actually specifies water temperature.
Has anyone else had a similar experience, or is fairly certain that this is/is not the problem?

Thanks for any help!

-Tato

#2 HomeBrew

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Posted 16 July 2010 - 05:36 PM

I have reconstituted many thousands of primers with room-temperature distilled water without any issues. I have also use primers thusly reconstituted years afterwards, also without problems. We store our primers at -20C. What were yours stored at?

If the primers worked two months ago, they should work now. If they don't, then it's more likely to be a template or cycling problem, or the water used was contaminated with a nuclease, or the primers were stored at the wrong temperature and underwent autohydrolysis (a supposed drawback of reconstituting in unbuffered water, but one I've not run into yet).

#3 perneseblue

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Posted 16 July 2010 - 08:53 PM

I have to agree with Homebrew. While I have only reconstituted a mere several hundreds of primers, I have never noticed any importance in temperature. Room temperature or even 68C works okay.

Primers are pretty tolerant and survive repeated heat thaw cycles relatively well. Autohydrolysis might occur but given the concentration of the primers, I have never noticed any different.

My only concern is nuclease contamination. Thus I dilute my stock primers in TE, to make a primer concentration of 100uM. Working stock primer of 10uM are made by further dilution with distilled water.

Even working primers in water survive well (3 year), despite repeated heat thaw cycles

I don't think the primer reconstitution is the problem. (unless you were using tap water, or there is an actual error in the primer, either in its synthesis or an order mix up).

It is more likely that the problem comes from the template. Perhaps the template was dirty. Perhaps the region which the primer anneals has nasty secondary structure (repeats, high GC content)
May your PCR products be long, your protocols short and your boss on holiday

#4 Tatooinedweller

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Posted 27 July 2010 - 04:18 AM

Thanks to both for the answers. The primers were reconstituted with nuclease-free water. I think they did some simple tests to see if it was the primer or the template, and the primers do seem to be the problem. Sadly, I'll probably never know what went wrong.




I have to agree with Homebrew. While I have only reconstituted a mere several hundreds of primers, I have never noticed any importance in temperature. Room temperature or even 68C works okay.

Primers are pretty tolerant and survive repeated heat thaw cycles relatively well. Autohydrolysis might occur but given the concentration of the primers, I have never noticed any different.

My only concern is nuclease contamination. Thus I dilute my stock primers in TE, to make a primer concentration of 100uM. Working stock primer of 10uM are made by further dilution with distilled water.

Even working primers in water survive well (3 year), despite repeated heat thaw cycles

I don't think the primer reconstitution is the problem. (unless you were using tap water, or there is an actual error in the primer, either in its synthesis or an order mix up).

It is more likely that the problem comes from the template. Perhaps the template was dirty. Perhaps the region which the primer anneals has nasty secondary structure (repeats, high GC content)



#5 gogreen

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Posted 27 July 2010 - 07:03 AM

Hi Tato, I also agree with Homebrew and Perneseblue, it should not be a problem to reconstitute primers in water at RT. The ice cold water idea doesn't sound very convincing to me either!. I resuspend my primers in TE buffer or Qiagen EB (10mM Tris Cl pH 8.5) at RT too! I make 100uM stocks and dilute them to a working conc of 10uM or 5 uM in water or EB.

(and is assuming they all don't--she's a post-doc and I'm the student, so I'll trust her on this one)

- I think that one doesn't have to trust everything said by people just because they are better qualified than them. Its good to think logically on things :)

But did you try the primers yourself again with some positive controls for primers and templates?

Edited by gogreen, 27 July 2010 - 07:04 AM.


#6 Maddie

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Posted 02 August 2010 - 08:19 AM

Is it a reverse primer? I ask because it's easy to make mistake when ordering a 3' to 5' reverse primer ;) .
Check the paperwork sent by AB and the sequence you sent them.
Have you tried this primer with a different 2nd primer? Maybe the 2 you have don't do well together.
If the reverse is the problem, try to test it with a different forward (or vis versa).

Good luck.

Maddie.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein




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