How primers can be measured? - (Dec/15/2008 )
How can i check primers concentration on agarose or Nanodrop?. The working primers are usually 10 uM.
i would use the nanodrop to determine the concentration.
I use a nanodrop to determine if the primers are contaminated, but I cannot accurately correlate concentration to absorbance with my primers-I think it's because each base absorbs the light at slightly different amounts, and the calculations the nanodrop uses is an average of the 4 bases. As primers are much shorter than genomic DNA, a high GC or AT content can throw off the calculations. I wouldn't trust it for concentration calculations but that's just me.
EDIT: I meant contaminated, not degraded...
How do you determine the degradation using a spec/nanodrop? They don't give you any information on that sort of thing, the 260:280 ratio works just fine with a pure dNTP solution.
The Nanodrop shows the working primer concentration is 98.18 ng/ul. How can i convert it to uM as working primer has?. I usually dilute primer to 10 uM for PCR reaction.
Sorry, I meant contaminated, not degraded... that shows how awake I am... Occasionally contaminates get into my primer solution (I'm not quite sure how...) and it gives me a funky looking curve. Whenever PCR doesn't work and the primers have a weird absorbance line, I just order new ones and the problem is solved most of the time.
The Nanodrop shows the working primer concentration is 98.18 ng/ul. How can i convert it to uM as working primer has?. I usually dilute primer to 10 uM for PCR reaction.
Doesn't the original tube say how many ng of DNA is present, as well as the number of nmol synthesised? Do you know what volume of buffer was added to the primer when it was resuspended initially?
If not, you'll just have to do things the hard way: calculations! You should know or be able to calculate the molecular mass of the oligo. Use that to go from ng/ul to uM.
Our standard procedure is to resuspend the oligo in 10x the number of nmol, which gives 100 Um stock. Others here will do that or something similar. Perhaps that's is what happened for you, but the documentation has been lost.