2 replies to this topic

[angel0071987]

Hi
I was hoping you could help - i seem to be going round and round in circles and getting more and more confused.

I have my primers (forward and reverse). I take the lyophilised primers and dilute/resuspend to a 100pmol/ul (=100uM) stock concentration.
I then make a primer mix of 10ul of F and 10ul R and 180ul water.
This is the working solution, of which i take 0.2ul for each 10ul reaction.


what concentration does that make the working solution of primer mix / in each well

is it a 1:20 dilution i.e 20pmol/ul  in the primer mix, which means in each well the conc is 2pmol??

Any help greatly appreciated. Thanks

[tea-test]

angel0071987, on 12 March 2012 - 01:43 AM, said:

Hi
I was hoping you could help - i seem to be going round and round in circles and getting more and more confused.

I have my primers (forward and reverse). I take the lyophilised primers and dilute/resuspend to a 100pmol/ul (=100uM) stock concentration.
I then make a primer mix of 10ul of F and 10ul R and 180ul water.
This is the working solution, of which i take 0.2ul for each 10ul reaction.


what concentration does that make the working solution of primer mix / in each well

is it a 1:20 dilution i.e 20pmol/ul  in the primer mix, which means in each well the conc is 2pmol??

Any help greatly appreciated. Thanks

Your stock is 100µM.

You dilute that 1:20 for your working solution for each primer, so your working dilution is 5µM (this 10+10µl and fill up to 200µl step).

In the reaction mix, this 5µM solution is again diluted 1:50 (0.2µl in 10µl). So you end up with a 0.1µM or 100nM concentration of each primer in your PCR reaction.

[tea-test]

angel0071987, on 12 March 2012 - 01:43 AM, said:

which means in each well the conc is 2pmol??

BTW, this a frequent mistake. 2pmol is NOT a concentration, it is the amount (number of molecules) of a substance.