Lambda DNA resists digestion
landel at helios
Thu Nov 3 15:03:30 EST 1994
In article <22313tcn-031194105635 at 18.104.22.168>,
Thomas Newman <22313tcn at ibm.cl.msu.edu> wrote:
>In article <396aas$ki1 at news.service.uci.edu>, rmauk at crick.bio.uci.edu (Rob
>> I have isolated lambda gt10 DNA by PEG precipitation followed by organic
>> extraction and ethanol precipitation, and find that it is almost
>> completely resistant to digestion by EcoRI and all other enzymes that I
>> have tested. Proteinase K treatment did not help, nor did additional
>> extractions and precipitations. Plasmid DNA digests normally with these
>> enzymes, but if the plasmid is mixed with the lambda DNA, it is rendered
>> undigestable. Any comments or suggestions regarding the nature of this
>> contaminant, or solutions to the problem would be greatly appreciated.
>> Thank you.
We have found that the following gives more consistently digestible DNA:
1) Do the PEG precipitation, resuspend phage in TE.
2) Extract the phage ppt with chloroform--this will remove PEG.
3) Add 10% SDS to make a final concentration of 0.5%
4) Heat to 65 deg
then carry on with the Prot K digest, and extract with phenol, then phenol/
chloroform, then chloroform, then EtOH precipitate.
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