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confirming concentration for primers in rt-pcr


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#1 Loris

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Posted 06 May 2009 - 05:45 AM

Hi,

Two questions:

1. I resuspend my primers at a concentration of 100 picomoles/microlitre. I need to convert to micromolar for my primers because you use lower concentrations of your primers in RT-PCR. I did the calculation and translated the 100 picomoles/microlitre to 100 u(mirco)M. Am I right? I can't help but second guess myself :)

2. What final concentration of primers do you usually use for RT-PCR?

Thanks!

#2 almost a doctor

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Posted 06 May 2009 - 06:11 AM

Hi,

Two questions:

1. I resuspend my primers at a concentration of 100 picomoles/microlitre. I need to convert to micromolar for my primers because you use lower concentrations of your primers in RT-PCR. I did the calculation and translated the 100 picomoles/microlitre to 100 u(mirco)M. Am I right? I can't help but second guess myself :)

2. What final concentration of primers do you usually use for RT-PCR?

Thanks!


Hi Loris, yes 100 picomoles/microliter = 100 micromolar (uM)
As for the concentration I use; it ranges from 200 to 400 nM (nanomolar). I usually dilute my 100uM stock to 10uM, and then use that for the PCR mix.

#3 Curtis

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Posted 09 May 2009 - 06:10 AM

Hi,

Two questions:

1. I resuspend my primers at a concentration of 100 picomoles/microlitre. I need to convert to micromolar for my primers because you use lower concentrations of your primers in RT-PCR. I did the calculation and translated the 100 picomoles/microlitre to 100 u(mirco)M. Am I right? I can't help but second guess myself B)

2. What final concentration of primers do you usually use for RT-PCR?

Thanks!


Hi Loris, yes 100 picomoles/microliter = 100 micromolar (uM)
As for the concentration I use; it ranges from 200 to 400 nM (nanomolar). I usually dilute my 100uM stock to 10uM, and then use that for the PCR mix.


It really depends, but for a normal conventional PCR I use 10uM working solution. this means I dilute my stock primers (100uM) ten times.

but I also use 100uM of primers sometimes, it really depends.

my Real-Time PCR primers are also 100uM stock and I top up to a final concentration of 10uM in the pcr reaction mix.




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