Hello! I want to use an old 100 mM concentrated primer for a PCR. There is probably some degradation due to time (6 years old). Thus I have quantified it in nanodrop so I could do a reliable dilution. However, nanodrop gives the concentration in ng/uL. For example, primer F was averaging 578.15 ng/uL. Since the primer is supposedly no longer 100 mM, I need to figure out how many ng/uL it should be in order to know how many ng/uL my diluted solution needs to be at 10 mM. Can anybody help me?
Here are the data of primer F:
MW = 6347.2
54.6 nmol
I did a simple calculation and got my 10 mM solution should be 63.47 ng/uL. However, I'm not sure about this.
I would not trust nanodrop readings to assess degradation. Nano will read the absorbance of all nucleic acids so even nucleotides and short degradation fragments will absorb and give you the impression that you have a good product. Oligos are very stable if dissolved in TE so if this oligo is for pcr then I would treat it as if it were fully intact and run a couple of pcrs with the primer at the expected dilution and at half that concentration and see if it works as a test of integrity. Primer is always in large excess in pcr so a small amount of degradation may not make much difference to the pcr working
In addition to Paul Rutland's answer, it seems to me that there must be a mistake in the concentrations you mention: you must mean µM and not mM: a 100 mM solution of your primer would amount to a concentration of 634.7 mg/ml. However, a 100 µM amounts to 6347 µg/ml or 634.7 ng/µl, close to your actual Nanodrop result of 578.5 ng/µl. Replacing mM by µM, a 10 µM solution is indeed 63.47 ng/ml.
When doing such calculations involving numbers with different orders of magnitude, it is of advantage to use the scientific notation with powers of 10.
Pierre Béguin Yes! µM** I am sorry. It was a careless error. Thank you for your reply. The same to Paul Rutland Thank you very much.
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