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Equimolar Mix Primer - (Oct/20/2013 )

Hi There

 

I'm a newbie  in this.

 

Currently I'm doing a PCR with 5 forward primer and 13 reverse primer.

My primers are in stock solution of 10uM

 

I need to put in an equimolar mixture of each primer to the PCR master Mix for a final concentration of 10pmol of each nucleotide in the pimer sets.

How do I do this?

 

Needhelp.....

TQ

-jpau-

So you are doing a multiplex PCR with 5 forward primers and 13 reverse which will give you a number of bands on a gel?

Without knowing the exact nucleotide mix in each of the primers your question is impossible. However, assuming that each base has an equivalent chance of being present in the sequence (i.e. all bases are present in equal amounts), then all you need to do is work out how much primer to add to get 10 pmol.

One way to do this is to work out how many pmol there are in 1 ml or 1 ul of a 10 mMol/litre solution...

-bob1-

Hi

 

TQ.

 

Well I guess it is multiplex PCR with 5 forward primer and 13 reverse primers.

Sorry  I guess I missed the info. Yes, each primers has an equivalent amount ( 20 bases )......

-jpau-

Ok, so how many pmol are there in 1 ul of a 10 umol/l solution?

-bob1-

= 10pmol/ul

 

I guess the calculation is as so....

 

1 ul of 10uM = 10umol/10-6L                   

                     = 0.00001 umol/ul

                     = 10 pmol/ul

 

Hope this is right?

-jpau-

Yes, that works - so can you now convert for each nucleotide?

-bob1-

Sorry, not quite sure what do you mean convert for each nucleotide? Sorry

-jpau-

In your original post you said:


I need to put in an equimolar mixture of each primer to the PCR master Mix for a final concentration of 10pmol of each nucleotide in the pimer sets.

How do I do this?

So I was trying to get you to work out how many of each nucleotide you would find in a 20 bp primer with equal proportions of A,T,C, and G...

-bob1-

Ehm, jpau, do you really have 18 different primers each of them 20 bp long, or you have just two of them?

 

Also, you said your primers were 10uM (that means 10umol/L or 10pmol/uL as you calculated). That is a concentration.

 

You said you need 10pmol of each in the reaction. 

That is technically not a concentration, concentration is amount/volume. So, either you need to have a concentration 10pmol/ul in your reaction and then it depends how big is your reaction volume. Or you need 10pmol (which is just an amount) in whatever the final reaction volume may be.

 

Difference is, for 10pmol in whatever volume you always take the same amount of stock. For final concentration 10pmol/ul in your reaction you need to calculate with the reaction volume (i.e for 20ul reaction you need 200pmol of primer, for 50ul reaction you need 500pmol of primer to get the same concentration).

-Trof-