Originally Posted by Buni
Hi I am a little confused and was hoping someone could help me.
I have a primer working stock of 10uM. For sequencing of a PCR product the requirement is for 3.2 pmol of primer and DNA template in a total volume (water) of 16ul.
I have worked out that I need to do a 1/3.125 dilution to get the right conc of primer - so this is 1ul of working stock primer and 2.125ul of water.
So then if I add 1ul of this primer dilution to 2ul of PCR product and 13ul of water will this be fulfilling the sequencing requirements?
Not sure what's going on here.
If you are setting up the sequencing PCR rxn yourself, you still need the reagents (PCR buffer, labeling dNTPs, Mg++, taq etc) those will make up the rest of the PCR total rxn volume.
If you are setting up template and primers and someone else will run it (like some sequencing core lab, etc) then you will turn over the DNA and primers to them, preferably in separate tubes.
If you are just calculating that primers and dna template need to be 16ul of volume for a sequencing rxn (say 25ul total rxn volume). Well that accounts for the actual seq. reagents and you are right, you got the right picomoles and dna temp. and the differenc is just water. So you would be right.