I did the protocol, but when quantifying said measuring at 260 nm the first purified and multiply by 3.3, which is the extinction coefficient however, I have as a control the concentration of the primers raw, but when doing the calculations, It does not give me the known values, also use a formula involving the characteristics of the sequence and did not give me the known concentration. Help!
After Purifying your Primers from polyacrylamide gel you then use nanodrop to measure it and use water or TE as blank.
Dear Cynthia,
I hope you are using the gel extraction kit to elute DNA from your gel. After which you can always read absorbance at 260 nm.
Abhishek
Hi Cynthia,
1. Weren't the DNA primers you purchased from a company already purified?
2. Please see this article at the link below. It gives an example of how to calculate the concentration of single-stranded DNA primers:
Title: How to Calculate a DNA Primer Concentration
Link: http://bitesizebio.com/9945/how-to-calculate-a-dna-primer-concentration/
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