Lambda DNA resists digestion

[Carlisle Landel]

In article <22313tcn-031194105635 at 35.8.197.53>,
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
>Mauk) wrote:
>
>> 
>> 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.

Good luck!

Carlisle Landel