Hi everyone,
I have extracted the human genome from whole blood and tried to digest 2 ugs of it with 20 units of Bcll enzyme. unfortunately, after some trials, I observed that most of the genome remained undigested. I prolonged the digestion time to 16 hours, but the result didn't change.
I tried another enzyme but the result was the same as before. I purified the DNA and eluted it with DDW ( to ensure that EDTA within TE buffer is not the reason) but the result was the same.
I studied somewhere that nucleosomes restrict restriction enzymes from accessing restriction sites. so this could be the issue? how can I remove the Histones (nucleosomes) from the DNA ? can I incubate the whole genome at 95 C and then digest it?
I assume that you are cutting at 50c with the BCl1.
Contaminants are many and you can test for them by cutting lambda dna in one tube and lambda with 0.5ug of your dna together in another tube. If the second lambda does not cut then you have contaminants present in your sample.
EDTA could be one ,excess protein bound to the dna or alcohol can all kill RE digestion/ One extra sample wash on the purification column may help or salt precipitation of the dna . Overnight incubation with 40 units of enzyme should not be a problem.
What is the OD260/280 ratio of your dna?
or maybe the sample was too much concentrated to allow enzymes to access the DNA and cut it.
try with lower concentrations, you could afterwards reconcentrate the sample by evapating some water.
for this new year, all the best
fred
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