When I measure the concentration and quality of the purified PCR product, the value of 260/280 is within the range of 1.8 - 2.2, while the value of 260/230 is always close to 0. Why is this happening? Can it present any complication for subsequent procedures?, such as performing another PCR from that purified product. And finally, would it affect nucleotide sequencing?
a zero ratio suggests that the absorbance at 230 is very high so the ratio is very low. This happens when you fail to wash away guanidinium salts in the dna binding buffer with enough wash buffer when using column dna purifications. Try loading a lower volume of dna or do a second wash step when purifying your dna. If you are doing re amplifications then you will use a very dilute dna sample so as to avoid over amplification so the salts will not interfere with the pcr. If the dna concentration is high then also only a small volume will be used in the sequencing mix and I would expect sequencing to work well
Thank you very much. So what would happen if you sequence like this with the absorbance close to 0?
The absorbance is not zero Isaac Falconi . The ratio of 260/230 is zero which means that either the a260 is zero or the A230 is huge. In your question you said that the ratio 260/280 is good (1.8) so the A260 cannot be zero so the A230 must be large so your dna is contaminated with some guanidine residue.. If your amplification was strong and the amount of dna needed for sequencing is small ( less than 4ul in a 20ul sequencing reaction) then I would expect the sequencing to work
Its true, you are right. I formulated my question wrong, it would be what would happen if it is sequenced if the ratio 260/230 is close to 0?
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