2 replies to this topic

[Loris]

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!

[almost a doctor]

Loris, on May 6 2009, 02:45 PM, said:

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.

[Curtis]

almost a doctor, on May 6 2009, 06:11 AM, said:

Loris, on May 6 2009, 02:45 PM, said:

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.

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.