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How much primer to add??

How much primer to add?? - PCR - Polymerase Chain Reaction Forum

How much primer to add?? - 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 02-18-2013, 12:44 PM
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Default How much primer to add??



Hi everyone
I have a 100然 Primer Stock which I'm going to dilute 1:10 giving me a 10然 working primer soln. I need 20pmol of primer for a PCR reaction that has a final volume of 100痞. Can't figure out how much primer to add. Can anyone help?
Thanks
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Old 02-22-2013, 04:45 PM
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Default Re: How much primer to add??

100然 corresponds to 100pmol/無, if you dilute it 1/10 then you can simply use 2無 per PCR.
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Old 03-02-2013, 05:12 PM
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Default Re: How much primer to add??

As Multiplexion has written, 100uM stock that you have got means 100pmol/uL and when you dilute it to 1:10, then your working concentration becomes 10pmol/uL. If you add 2uL it means you have added 20pmol/uL of primer to a reaction of 100uL.
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Old 03-28-2013, 06:15 AM
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Default Re: How much primer to add??

Hi, its very simple, here is the calculation

you have 100uM stock = 100pmols/uL, you did 1:10 dilution so stock concentration became 10uM = 10pmols/uL;

Intial concentration (C1) = 10uM = 10pmols/uL
how much to take (V1) = ??
Final concentration (C2) = 20pmols/100uL = 0.2pmols/uL
Final Volume (V2) = 100uL

C1*V1 = C2*V2

10*V1 = 0.2*100
V1 = (0.2*100) / 10

V1 = 2 uL

Hope you understood.
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