A PCR protocol requires 20 picomoles of forward primer in a volume of 1 L and 20

Question

A PCR protocol requires 20 picomoles of forward primer in a volume of 1 L and 20 picomoles of reverse primer in a volume of 1 L as well. Primers were ordered from IDT and the manufacturer provides the primers in lyophilized form, therefore they need to be reconstituted in TE buffer. According to the printed labels on the primer vials, there were 93.8 nmoles of forward primer, and 134.3 nmoles of reverse primer. To reconstitute the primers: 825 L of TE buffer to forward primer and 1125 L of TE buffer to reverse primer.

(a) What is the concentration of the two primers in pmoles/L in stock forward primer and stock reverse primer?

(b) In what proportion should stock of forward primer and reverse primer should be added to the TE buffer to attain 20 pmoles/L concentration? Show your calculations.

Explanation / Answer

Answer:

a. Forward primer = 93.8 nmoles, Volume = 825 L of TE buffer

Forward primer concentration = 93.8/825 = 0.11369 nmoles/L = 113.69 pmoles/L OR M.

Reverse primer = 134.3 nmoles, Volume = 1125 L of TE buffer

Forward primer concentration = 134.3/1125 = 0.11938 nmoles/L = 119.38 pmoles/L OR M.

b. Primer dilution in TE buffer:

(It is not clear from the question whether a single dilution is to be prepared with both forward and reverse primers at 20 pmoles/L OR they are to be diluted separately to 20 pmoles/L. The calculation below has been done taking into account that it has to be done separately, since this is the normal protocol followed for routine molecular biology lab work)

Forward: (Final volume say 10 L)

Initial concentration = 113.69 pmoles/L

Using V1S1 = V2S2

V1* 113.69 pmoles/L = 10 * 20 pmoles/L

V1 = 1.759 L

So, 1.759 L of forward primer from the stock should be added to 8.241 L of TE buffer to get a final concentration of 20 pmoles/L.

Reverse:

(Final volume say 10 L )

Initial concentration = 119.38 pmoles/L

Using V1S1 = V2S2

V1* 119.38 pmoles/L = 10 * 20 pmoles/L

V1 = 1.675 L

So, 1.675 L of forward primer from the stock should be added to 8.325 L of TE buffer to a final concentration of 20 pmoles/L.

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