standard curve - (Oct/23/2009 )

Hi,

I am trying to optimize my primers and probes to check for the viral copy number. I have used the whole viral DNA of know copy number to make my standards. I nanodroped the viral DNA sample and I got 70.8ng/ul concentration. I calculated the copy number using a software,which gave me 5.05*108 copy number. My primers were of 10nmol concentration. I added 1ml to it and made it to 10umol concentration. I diluted it down to 3umol. Then I used it in the reaction. My probe concentration was 100um. I made 25um stock and diluted it further down to 2.5 um concentration and then I used it in the reaction. I am using ABI 7000 instrument. My amplification is not very good. I am not sure how to go about it optimizing it. Can somebody help me out with this?

Regards
Gladis

gladis on Oct 23 2009, 08:16 PM said:

There you go!!!
By adding 1 ml to a 10nmol conc primer you are diluting it further and not concentrating. So it cannot become 10 umol but it s 10 pmol now.. so essentially u have added less primers in your reaction.. may the the reason for no good amplification...

BEst luck!!

I fear you may be confusing amounts with concentrations. I think what you meant was that you received 10 nmol of primer (an amount!) and diluted it to 10 umolar (a concentration!) by adding 1 ml of buffer.

But the real problem is probably that you are using too much template. Try the reaction with 100x or 1000x smaller amounts of the viral DNA.