I have almost the same query. I have extracted the DNA from old blood samples. The nanodrop concentration of the extracted DNA is also very low, ranging from 5 ng/ul to 50 ng/ul. I have to perform nested PCR. I have performed the reaction by using 100% extracted DNA, i.e., without dilution. The PCR showed very good results on the sample having the nanodrop concentration of 7.89 ng/ul, and I used it at 5 ul in a 25 ul reaction. How can I calculate the same concentration for other samples having different nanodrop concentrations? Also guide me, please, if there is any need to make dilutions as running solution for all the samples having low concentrations or not. Also guide me, please, if there is any need to make the dilutions for the samples having nanodrop concentrations above 30 or 40 or 50, to use it as 100% with relatively high nanodrop concentrations Because if we use it without dilution, the sample taken will be less than 1 ul, like 0.3 ul, 0.4 ul, or 0.5 ul, which are very low and may result in negative results. If we need to make dilutions for these samples, then please guide me on how I will make dilutions.
Make some aliquot of 50 ng/ul DNA, and dilute the aliquot instead of full volume.
Not only should you consider the DNA concentration measured by Nanodrop, but you should also pay attention to its purity (A260/A280 ratio). Secondly, in your 25 µl reaction, you used approximately 40 ng of DNA. You can easily adjust the DNA amount in your samples to match this quantity.
For example, for a sample with 50 ng/µl:
Divide 50 by 7.89 (≈6.3). This means 1 µl of 50 ng/µl DNA is equivalent to 6.3 µl of 7.89 ng/µl DNA.
Dilute 1 µl of 50 ng/µl DNA with 5.34 µl of DNase-free water or TE buffer.
Use 5 µl of this diluted DNA for your PCR reaction.
If the DNA concentration is too high, you may observe smearing instead of clear bands.
- Badges
- Science topic
- Similar topics
- Instrumentation Engineering
- Instruments