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resuspend Primers in TE or in water? - (Jul/31/2008 )

Hi,

I have a simple question. Can anybody explain me the difference between ressuspend liofilized primers in T.E. or in water??

Thank´s

-chicoc-

QUOTE (chicoc @ Jul 31 2008, 07:56 AM)
Hi,

I have a simple question. Can anybody explain me the difference between ressuspend liofilized primers in T.E. or in water??

Thank´s


I normally use water, but I doubt that it makes much difference.

-smu2-

probably not in short term. But in long term storage welll... that is a bit unknown.

Autocalve water is acidic from the disolved CO2. And an acidic solution is probably not the best thingyou want to keep your primer stocks in for long term storage (years to decades). Thus I prefer TE.

Of course, my working primer solution is made by diluting my stock primers in TE with sterile distilled water.

-perneseblue-

The point of the EDTA is to chelate Mg2+ ions. The idea is that most DNAses require Mg2+ ions as a cofactor, and thus will have no catalytic activity if all of the magnesium is bound up by the EDTA. It seems to me that most DNAses work on dsDNA anyway, so would they have little effect on single-stranded primers to begin with. The Tris buffer is theoretically a better storage medium than water as discussed above, but, empirically, I've always resuspended my primers in sterile water, and never had any problems with them.

-HomeBrew-

QUOTE (HomeBrew @ Aug 1 2008, 04:14 AM)
The point of the EDTA is to chelate Mg2+ ions. The idea is that most DNAses require Mg2+ ions as a cofactor, and thus will have no catalytic activity if all of the magnesium is bound up by the EDTA. It seems to me that most DNAses work on dsDNA anyway, so would they have little effect on single-stranded primers to begin with. The Tris buffer is theoretically a better storage medium than water as discussed above, but, empirically, I've always resuspended my primers in sterile water, and never had any problems with them.


If you are using Taq: Taq requires Mg2+ to work , so using TE (to dilute primer or resuspend DNA) you have to increase the Mg concentration in your PCR reaction! (1mM EDTA = an extra 1mM Mg2+)

-gebirgsziege-

QUOTE (gebirgsziege @ Aug 1 2008, 01:50 PM)
QUOTE (HomeBrew @ Aug 1 2008, 04:14 AM)
The point of the EDTA is to chelate Mg2+ ions. The idea is that most DNAses require Mg2+ ions as a cofactor, and thus will have no catalytic activity if all of the magnesium is bound up by the EDTA. It seems to me that most DNAses work on dsDNA anyway, so would they have little effect on single-stranded primers to begin with. The Tris buffer is theoretically a better storage medium than water as discussed above, but, empirically, I've always resuspended my primers in sterile water, and never had any problems with them.


If you are using Taq: Taq requires Mg2+ to work , so using TE (to dilute primer or resuspend DNA) you have to increase the Mg concentration in your PCR reaction! (1mM EDTA = an extra 1mM Mg2+)


i just got primer from company and in the note 1xTBE is suggested to used.
do any of u know the different?
malmama

-malmama-

I prepare the stocks in TE 1X (usually 100µL). For normal PCR I prepare a "working solution" and dilute the primers in TE, but for QPCR I prepare the working solutions in water (just prepare less quantity) because high concentrations of salts inhibits the reactions.

-merlav-

biggrin.gif Hi, I resuspend my primers in water because I use them to sequencing and the T.E. kills the enzymes during the sequencing reactions.

I recommend you always do aliquots of you primers to avoid frequent frozen-defrost, that could cause primer degradation.

-u01dfd7-