I wonder if there is a possibility to perform purity and quantity measurement of PCR product after ExoSap treatment?
What about blank sample in this case?
PS I think about 260/280 and 260/260 ratios for purity measurement and DNA quantification on NanoDrop spectrophotometer.
I am not sure what kind of blank sample would be proper - after PCR and ExoSap treatment.
ExoSap contains exonucleas and alkaline phosphatase. So, protein concentration (260nm) will be high and unincorporated nucleotides and digested primers will contribute to DNA concentration. Therefore, it will not be accurate using Nanodrop on top of inaccuracy often caused by Nanodrop itself.
- blank sample - inactivated alkaline phosphatase and exonuclease in PCR mixture
- test sample - inactivated alkaline phosphatase and exonuclease in PCR mixture with clean PCR product
this setting will probably be reasonable for reliable measurement.
Unfortunately, in is impossible to perform any measurement of dsDNA dissolved in PCR mixture cleaned wit use of ExoSap. greetings.
even if this is too late. I just tried to measure DNA after an ExoSAP purification of PCR products. I used a negative control (inactivated enzymes, buffer and water) as a blank and it is NOT working. The wavelength ratios are out of normal range. I agree. Measure the Product before purification (but here I am not sure how precise the results will be) or talk to the sequencing company. In my former labs, we never measured the PCR product quantity for sequencing, and we did the sequencing by ourself (Sanger Sequencing).
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