MolBio: Troubleshooting ExoIII Nested Deletion Creation

Sandy Kielland (kielland@UVic.CA)
Mon, 13 Jul 1998 11:56:45 -0700

I'm currently trying to create a nested deletion library of 4.5kb and 6kb
clones. I'm using a the Pharmacia kit that uses Exonuclease III for the
single-stranded digestion followed by cleanup with S1 nuclease.

I'm running into trouble with the ExoIII digestion. I originally prepared my
plasmid DNA sample using a Qiagen Mega prep and CsCl gradient purified the
samples as well. I am wondering if maybe this was too much manipulation for
the samples and may have introduced nicks in the DNA that allow the ExoIII
to digest from multiple sites. ExoIII digestion leads to multiple products
when analyzed by gel even after only 3 min of digestion.

Originally I thought I had not protected the vector well enough with a 3'
overhang. But I have since sequentially digested the sample with Sac I to
protect the vector from ExoIII followed by EcoRI digestion to produce the
ExoIII susceptible end on the insert.

Has anyone had similar problems?

Thanks,
Mike (icarus@uvic.ca)