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EDTA - real time PCR (Jun/30/2005 )

The protocol: Add 2 ┬ÁL of 0.5 M EDTA to each cDNA reaction. Store the cDNA samples at 4˚C for one month.


When I use this cDNA sample, do I have to get rid of EDTA first or is that okey if I use the sample directly for real time PCR? If i have to get rid of EDTA. How can i do it?

-indoubt-

You should be able to use it directly for PCR, however, this will depend on the volume of your cDNA reaction... if it is small (10-20 microliters) and you added 2 uL EDTA, then this could affect your PCR. To fix this you could add some Mg to the PCR mix, you would need to titrate it to find the most effective concentration.

BTW, I have never added EDTA to a cDNA rxn, but I store mine at -20 or -70.

Bob

-bob1-

QUOTE (bob1 @ Jun 30 2005, 09:56 PM)
You should be able to use it directly for PCR, however, this will depend on the volume of your cDNA reaction... if it is small (10-20 microliters) and you added 2 uL EDTA, then this could affect your PCR.  To fix this you could add some Mg to the PCR mix, you would need to titrate it to find the most effective concentration.

BTW, I have never added EDTA to a cDNA rxn, but I store mine at -20 or -70.

Bob


Why should i add MG++? What role does it have in presence of EDTA? So for storing of cDNA sample you use sterile water and store them at -20 or -70? How long can you store the sample at this temperature?

-indoubt-

EDTA chelates the Mg++ thus taking the EDTA out of the reaction and freeing up the divalent cations in the pcr rxn to do what they need to do.

So far as I am concerned cDNA can be stored at -80 almost indefinitly... at -20 for at least a couple of months.

-pBluescript-