2 replies to this topic

[tsigoloib]

Hello there,

I have a forward and reverse primer which I made to 100uM concentration separately. Now for my PCR, I want to make 0.4uM final concentration in a 100 ul reaction. The primer length is 72 bases for reverse and 43 bases for forward. How do I dilute it?
Thanks

[Prep!]

tsigoloib, on Mar 17 2010, 11:44 AM, said:

Hello there,

I have a forward and reverse primer which I made to 100uM concentration separately. Now for my PCR, I want to make 0.4uM final concentration in a 100 ul reaction. The primer length is 72 bases for reverse and 43 bases for forward. How do I dilute it?
Thanks

Length has no role here...
N1V1 = N2V2
N1 = 100 uM (your stock)
V1 = the colume in question to be aliquoted to prepare 100 ul 0.4uM conc
N2 = 0.4 uM
V2 = 100 ul

So,
100*? = 0.4*100
? = 0.4*100/100
? = 0.4 uL

So u add 0.4 ul and make up the volume to 100 ul (after also adding the other reagents, like the template, buffer, taq etc)

I hope this is clear...
If you dont have the facility to aliquot 0.4 ul, the dilute the stock 4 times or 8 times and then take a higher aliquot!!!
Good luck!!!

Edited by Pradeep Iyer, 17 March 2010 - 04:03 AM.

[tsigoloib]

Hello Pradeep,
I have a related question. I have 25 ul of a PCR product of 177 bps at a concentration of 200ng/ul. This means that I have 5 ug of DNA in a tube. Now how do i calculate the number of fragments in this? I understand that it weights about 660g/mole per base pair. So accordingly, 177bps times 660g/mole per bp=1.16X10 5 g/ mole.

Now the point is I have to do a nested PCR using the above PCR product such that my template has ~800 million fragments. So how many micro litres do I need to take from the above PCR product to use as template in my nested PCR? I would appreciate any help in this.

Thanks
Franco

Pradeep Iyer, on Mar 17 2010, 08:03 AM, said:

tsigoloib, on Mar 17 2010, 11:44 AM, said:

Hello there,

I have a forward and reverse primer which I made to 100uM concentration separately. Now for my PCR, I want to make 0.4uM final concentration in a 100 ul reaction. The primer length is 72 bases for reverse and 43 bases for forward. How do I dilute it?
Thanks

Length has no role here...
N1V1 = N2V2
N1 = 100 uM (your stock)
V1 = the colume in question to be aliquoted to prepare 100 ul 0.4uM conc
N2 = 0.4 uM
V2 = 100 ul

So,
100*? = 0.4*100
? = 0.4*100/100
? = 0.4 uL

So u add 0.4 ul and make up the volume to 100 ul (after also adding the other reagents, like the template, buffer, taq etc)

I hope this is clear...
If you dont have the facility to aliquot 0.4 ul, the dilute the stock 4 times or 8 times and then take a higher aliquot!!!
Good luck!!!

Edited by tsigoloib, 17 March 2010 - 09:50 PM.