We would like to digest genomic DNA.
Our restriction enzymes APE K1 and BAM H1 will cut lambda DNA, but will not cut our genomic DNA samples, even if we clean them up with several methods after isolation (Ampure bead purification with ethanol washes; Ethanol-sodium acetate precipitation). On an agarose gel, we always see a large fraction of genomic DNA that remains undigested, if we compare against an undigested sample only a very small amount of the sample has been digested. We are digesting for 8 hrs, at the recommended temperatures, with recommended buffers by NEB and with a large enough amount of enzyme.
We have tried various protocols to extract genomic DNA but the digestion problem persists with phenol-chloroform and silica-based protocols (including one using CTAB lysis buffer).
If we mix lambda DNA with the genomic DNA, the enzyme won't cut the lambda DNA nor the genomic DNA, so we concluded that inhibitors prevent the digestion reaction.
The DNA is excellent for PCR-based approaches, but restriction digests do not work properly.
The species we are working with is a lichen (fungus+Nostoc) and polysaccharides and secondary compounds could be a problem.
Has anyone managed to get rid of inhibitors which are co-precipitated (and co-isolated) with genomic DNA so that the DNA can be digested?
Advice is highly appreciated!
Yes, polysaccharides are hard to purify. Try using Lyticase and/or Qiagen agarose gel purification kit . I would also suggest washing with 50 % EtOH instead of 70%. Hope that helps.
We solved this problem by using another enzyme. In fact, we tried 12 out of which two worked perfectly well. We will use the digested DNA for RAD tag sequencing.
The DNA extraction method didn't matter in the end. Both samples extracted with chloroform phenol and with silica membranes worked. The enzyme made all the difference! ApeK1 just would not cut our DNA properly, nor would BAM H1. But SAU3a and Hind 3 worked just fine with samples extracted in both ways.
We used Silica membranes, CTAB buffer for lysis and guanidium hypochloride in ethanol as a binding buffer, 70% ethanol as wash buffer, and eluted the DNA with AE buffer. I have posted the protocol in another one of my Q&As. Link is here: https://www.researchgate.net/post/How_to_optimize_DNA_binding_to_silica_columns/1
Thank you. I think the DNA extraction problem is solved by CTAB method. There are still problem about the digestion. What's the ratio between the enzyme and DNA in your system? I use 2 ul enzyme(100,000units/ml) for 8 ug DNA in 20 ul system.Some of the DNA is digested,but some are not. It is strange , is it?
Have your enzymes gone bad perhaps? Try a new batch...
I think for us it was some property of the DNA that made these particular enzymes not cut. The other enzymes we ended up using cut the same DNA extractions perfectly...
- Badges
- Science method