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primer conc for sequencing

primer conc for sequencing - PCR - Polymerase Chain Reaction Forum

primer conc for sequencing - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 03-24-2010, 03:14 AM
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Post primer conc for sequencing



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?

Thanks.
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Old 03-24-2010, 04:50 PM
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Post Re: primer conc for sequencing

Quote:
Originally Posted by Buni View Post
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?

Thanks.
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.
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Old 03-25-2010, 06:59 AM
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Default Re: primer conc for sequencing

Quote:
1uM = 10e-6 moles/liter x 10[-6] liters/ul = 10e-12 moles/ul = 1 pmole/ul
source : [Only registered users see links. ]
thus 3.2pmol/ul = 3.2uM (so u only need to put 1ul per reaction tube)
from M1V1=M2V2
V1=M2V2/M1=0.32V2 (1/3.125 also the same) but sometimes u may require more than 1 reaction for different samples thus you can make more V2.

Note: for PCR product please check and make sure within the range, 1-3ng for 100bp till <500bp something like that. As long as your 2ul does not go overboard too much should be good.

strange your final volume is 16ul. but heck its just me i guess
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