I am trying to get isolated bands from restriction digestion of a large plant genome (>800Mbp). After discussing with the people in my lab, their experience seems to have got a smear instead of bands which is expected when cutting such a large genome.
Any good idea?
What I could think of :
1) rare cutter
2) successive cut (i.e. cut -> fraction the smear -> cut)
Thanks in advance.
Depends, why do you need it. If you need sharp bands to be isolated, rare cutter is an option. To avoid smearing, you need to prevent DNA shearing - ie no vortexing, pipetting etc. You should isolate and restrict your DNA in situ - it means, get your cells into a small block of agarose (low %) - treat with all you need for isolation and restriction (buffers and enzymes will diffuse there). Then, put this block inside the well in agarose gel and separate (use pulsed field elfo for large fragments).
I think in this case, best option is to develop BAC clones and then identify desired BAC clones by Southern/other screening techniques. As you will always get smear when you will digest with good restriction enzymes or if you use rare cutters then size of fragment will be large and visualization on gel will be difficult.
I f you really want to do so (although i never have seen an application were it really was necessary (such a genomic library is generally the better choice) you will need to extract nuclear DNA with a ultracentrifuge. Otherwise you will always run into the shearing problem.
Actually, I am trying to compare the similarities of two genomes in general.
The background is I am using a k-mer subtraction method, by which specific 30-mers of two genomes are extracted from whole-genome sequencing reads, to identity mutation between the genomes. To ensure such kind of k-mer subtraction method is feasible, I have to know if the genomes is similar enough that I would not get tons of noise (i.e. specific k-mers but not the point of mutation).
Yesterday we have derived some dry experiments for me to check for the similarities.
Thank you for the answers, certainly eye-opening.
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