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Real Time PCR dilution

Real Time PCR dilution - Real-Time PCR and Quantitative PCR Forum

Real Time PCR dilution - Real-Time PCR and Quantitative PCR Forum


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  #1  
Old 12-19-2010, 07:21 AM
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Default Real Time PCR dilution



Hello,

I am new to Real time PCR work. My custom synthesized primer stock conc is 10nM (nanomoles) and would like to make my working stock conc of 300nMolar (nanomolar). can some one help me out to make my working conc.

Thanks in advance
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Old 12-19-2010, 01:28 PM
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Default Re: Real Time PCR dilution

This is an easy calculation if you use the formula

C1*V1 = C2*V2

where C is concentration in nM
and V is volume in microlitres or nanolitres

Also see this:

PCR Primer Dilution

Last edited by admin; 12-19-2010 at 02:20 PM.
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Old 12-20-2010, 06:21 AM
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Default Re: Real Time PCR dilution

thanks for message.
My primers for real time PCR stock conc/Amount is 10nmol and I want make working conc for 300nM. I guess C1V1=C2V2 will not work here because my stock conc is in nMOl and working should be in nanoMolar

My probe stock conc is 100 pmol/microlitre and want to make working conc for 100nM. please guide me in this regard

thanks in advance
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Old 12-20-2010, 11:30 AM
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Default Re: Real Time PCR dilution

Hi you can still do this by using a primer molecular weight calculator, using your sequence (input GCAT sequence) then you can calculate your needed concentration from the moles and calculated molecular weight.
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Old 12-22-2010, 02:21 PM
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Default Re: Real Time PCR dilution

I am confused about your working stock conc that primer was at 300nmol and probe at 100nmol, most working stock is from 5Ám/Ál to 40Ám/ul, did you mean the final primer/probe reaction conc( add 0.5ul primer or probe to 25ul final reaction), if so, you should make primer working conc as 15Ám/ul and probe as 5Ám/ul,
add 100Ál water to your original stock conc primer (10nmol) to gain 100Ámol Further Stock Conc (FSC) primer, then dilute 15Ál FSC primer into 85Ál water to gain 15Ám/ul.
dilute your stock conc probe as 20 times to gain 5Ám/ul working conc probe.

Last edited by namishu95; 12-22-2010 at 03:59 PM.
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