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How long we can keep Diluted Primer? - (May/21/2007 )

hi friends,

Yesterday, I diluted my stock primer (100uM) to 5X and 10X working concentration using PCR grade water. Today, I runned PCR using these dilutions but I have no product. I checked on Biolabs' (my primer sapplier) web site about it but I found nothing.

would you please tell me how long diluted primers can be keept at -20 oC.

Thanks!

-Doyo-

QUOTE (Doyo @ May 22 2007, 07:11 AM)
hi friends,

Yesterday, I diluted my stock primer (100uM) to 5X and 10X working concentration using PCR grade water. Today, I runned PCR using these dilutions but I have no product. I checked on Biolabs' (my primer sapplier) web site about it but I found nothing.

would you please tell me how long diluted primers can be keept at -20 oC.

Thanks!


It all depends on how clean you work. If you take care and don't contaminate the primer solution with nucleases, it can last 2 years and probably more.

If there is a problem with the PCR, a general rule of the thumb says that the problem comes from the template. Diluting your template often helps (1:10 to 1:50), as even the cleanest genomic DNA is often contaminated with junk that will inhibit the PCR reaction. If your template is plasmid DNA, perhaps it is too concentrated.

And from the sounds of your post, it seems to be early days in the PCR. Have you tried optimising the reaction's conditions? Perhaps lowering the annealing temperature.

-perneseblue-

in my lab the primers are stored in -20c for years....
perneseblue gave some good suggestion, try to be more sure about DNA purity and amount

-T. reesei-

QUOTE (T. reesei @ May 22 2007, 04:46 PM)
in my lab the primers are stored in -20c for years....
perneseblue gave some good suggestion, try to be more sure about DNA purity and amount



Ya thanks a lot now I have very beautiful band.

Priviously I think I did mistake on pipating the primers.



Peace!

-Doyo-

You can check the primer integrity on a gel. Just to be sure. But primer degradation is the last thing comin to my mind in such cases... wink.gif

-kr├╝melmonster-

We make new primer dilutions from stock every time.

-scolix-