sheared DNA/nuclease inactivation

John H McDonald mcdonald at ravel.udel.edu
Wed Mar 24 14:51:01 EST 1993


Does the mechanical shearing of DNA that occurs in a normal DNA prep
produce only blunt-ended DNA, or does it also produce some sticky ends?  I
ask because I'd like to ligate an adapter on to restriction fragments, and
I want to be very sure it doesn't get ligated anywhere else.  
    If my DNA prep does contain some sticky ends, I'd like to do the
following: 1) Treat with mung bean nuclease or S1 nuclease, to blunt the
ends. 2) Do a restriction digest.  3) Do a partial fill-in reaction, to
keep fragments from sticking to each other in the next step.  4) Ligate on
the adapter. 5) PCR amplify the fragments.    I obviously need to
inactivate the nuclease after step 1, but phenol/chloroform extraction,
precipitating, resuspending could create more sticky ends.  Everything
I've read about mung bean and S1 nuclease says they can't be heat
inactivated.  Is this true, or does it just take a little longer or a
little higher temperature than other enzymes?  Is there another enzyme,
one that can be heat-inactivated, that I could use to blunt any sticky
ends?



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