We have a PCR amplicon which is around 10kb (plasmid backbone). We have to digest it with DpnI before proceeding with other steps. The gel extraction concentration is generally 1ug-1.5ug per 100ul PCR reaction. However, on DpnI digestion, the concentration drops to around 400ng. This is too low for our requirement. Does DNA conc. drop this drastically upon DpnI digestion? How do I improve the concetration? Thanks in advance:)
Hello Manali,
you can easy increase concentration of restricted DNA (after DpnI cleavage) by ethanol precipitation:
https://openwetware.org/wiki/Ethanol_precipitation_of_nucleic_acids
Hope this help
I'm assuming most of the drop in concentration is due to the fact that most of the volume of your restriction digest is buffer. You can set up more than one digest, maximize the amount of DNA in your digest, or use a spin-vac to evaporate your digested DNA to reduce the volume (and increase the concentration of DNA). Good luck!
When measuring DNA on a spectrometer it is very important to zero the spectrometer with exactly the same solvent that the dna is dissolved in. The amount of dna should not vary after digestion but incorrect zeroing may give unexpected results
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