help regarding primers reconstitution - (Apr/18/2005 )
hi
well i have this question abt primer reconstitution lingering in mind for the past few days...some literature says that lyophilized primers should be dissolved in Tris-EDTA buffer. some says only10 mM tris should be used....
i would really appreciate if someone could guide me...whether to used tris-edta or only tris. and their required pH also
thanks in advance..
swarup.. 
Hi Swarup,
I usually just reconstitute my primers in sterile water for use in PCR etc. and freeze aliquots (avoid freeze/thaw). If you like you could use 10mM Tris and this would probably make the primers a little happier but I have never had much of a problem with standard primers with that respect.
Totally avoid using Tris-EDTA the EDTA will chelate the Mg which polymerases absolutely require for activity.
Hope this helps,
Scott
I usually just reconstitute my primers in sterile water for use in PCR etc. and freeze aliquots (avoid freeze/thaw). If you like you could use 10mM Tris and this would probably make the primers a little happier but I have never had much of a problem with standard primers with that respect.
Totally avoid using Tris-EDTA the EDTA will chelate the Mg which polymerases absolutely require for activity.
Hope this helps,
Scott
hi scott..
thank you for ur advice...but just another query....does every freeze/thaw cycle affect ur primer?....can i make a working stock of 500 pmoles and then later dilute it to 50 pmole during every pcr or should i make aliquots of 50 pmoles (though tedious)?....which is be better?...
thanks again...in advance
swarup
Freeze thawing does affect the quality of your primers, especially if they are in water.
Scott is right about diluting in TE as EDTA will chealate Mg ions within the PCR reaction. However, I dilute my primers in TE to a stock concentration of 250uM and then make working stocks with water. The dilution to the working stocks with water (10uM) and then further dilution into the PCR reaction, the amount of TOTAL EDTA in the PCR reaction will be neglible and won't affect the reaction.
Diluting primers in TE is ideal as the primers are happy, however you must take scotts point into account.
Cheers
nick
Hi swarup,
Nick is absolutely right in terms of as long as the subsequent dilutions are in water then the EDTA in TE won't effect the subsequent reactions. However I have never had a problem with my stock primers.
When I get a primer in I reconstitute it in water to 100uM stock and freeze this in 3-4 tubes, at that time I will also make up a working stock at 10uM by diluting the stock 1 in 10. I don't tend to worry too much about freeze thawing the working dilution too much (I'll use it 5 or 6 times with freeze/thawing even more), if the PCR stops working than I will simply take one of my stock and re-dilute to create a fresh working dilution.
The thing to avoid is freeze/thawing your stock too much. As Nick suggests TE may be beneficial in your stock to increase primer stability. However, I haven't found any problems with my stock solution going 'off' in water as I will only ever freeze thaw a couple of times each tube.
Hope this helps.
Scott