I had gel eluted a PCR product whose concentration according to Nanodrop was 16.5ng/ul. 20ul of this PCR product was treated with 0.5ul of FD Dpn1. Hence, the yield of PCR product in this 20.5ul reaction mixture was calculated to be 16ng/ul.
Now, 1.53ul(1.23ng/ul) was used to treat with T4 PNK(0.4ul) in a total reaction mixture of 20ul. PNK was then purified using spin filter and column and DNA was eluted using 15ul of Elution buffer.
Reading on nanodrop is showing A260/A280 of 4, which does not indicate the presence of DNA. Hence, How do I calculate the concentration of DNA after PNK treatment?
First off, it's worth noting that an A260/A280 ratio of 4 is definitely unusual and suggests that there might be some sort of contamination or interference with your sample. Typically, you'd expect a ratio of around 1.8 for pure DNA, but there are a number of factors that can affect this value.
That being said, let's focus on calculating the concentration of DNA after your PNK treatment. If you know the initial concentration of your PCR product (16ng/ul), you can use that to estimate the amount of DNA present in the 1.53ul of PCR product you used for your PNK reaction.
So, if 20ul of your PCR product had a concentration of 16ng/ul, that means the total amount of DNA in that reaction was 320ng (16ng/ul x 20ul = 320ng). If you took 1.53ul of that reaction mixture for your PNK treatment, you would have 1.53ul x 16ng/ul = 24.48ng of DNA in that sample.
Now, after treating that sample with PNK and purifying it, you eluted the DNA in 15ul of elution buffer. If you assume that all of the DNA from your PNK reaction was recovered in that elution, then you can calculate the concentration of your final DNA sample by dividing the amount of DNA by the volume of the elution.
In this case, your 1.53ul of PCR product was diluted 13.07-fold (20ul total reaction volume - 1.53ul of PCR product used = 18.47ul of other reaction components; 15ul elution buffer / 18.47ul reaction components = 0.8107 or a 13.07-fold dilution). So, to get the final concentration of DNA in your elution, you need to multiply the amount of DNA you calculated above (24.48ng) by 13.07 to correct for the dilution.
When you do this, you get a final concentration of 319.6ng/ul, which is pretty close to your initial concentration of PCR product. However, keep in mind that the unusual A260/A280 ratio suggests that there may be something going on with your sample that is interfering with the Nanodrop readings. So while this calculation can give you an estimate of your DNA concentration, you may want to consider other methods of quantification or further investigation to confirm your results.
High OD260/280 ratios are usually caused by guanidine salts used to denature and bind the dna being purified. You could wash the column twice after binding before eluting the gel purified dna and then measure the ratio again to make sure that the amount of dna is correct for the pnk reaction
I think the problem is that you are starting with too little DNA. You only used about 1ng DNA in your reaction (and that assumes the initial nano drop concentration was correct and 100% of that reading was the purified DNA). I believe the efficiency of recovery of such tiny quantities of DNA is low and you probably ended up with virtually no DNA at the end. Try increasing your DNA amount to at least 10ng if not 100.
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