I do not know what the solution to your problem is. I do not have first
hand information that fits your situation. However, you can use me for
bouncing ideas.
Exo III is usually well behaved and a precise tool, so you may be right to
suspect the plasmid DNA. I do not know why the decision to use both the
Qiagen Mega prep and the CsCl gradient method. Either method, if done
correctly, is more than able to give you unnicked DNA useful in the Exo III
procedure. However, was your plasmid DNA ever stored unbuffered? DNA at
appreciable concentrations is acidic in water, and will give you
depurination upon storage in water. Exo III is also an apurinic
endonuclease in the presence of Mg, with the APendo activity about 10% as
strong as the exo activity, bond broken for bond broken. Depurinated DNA is
not changed in gel mobility, so you need to break the APsite backbone to
see the problem. One way is to use exo III in the presence of 5 mM Ca++
without Mg. Under this condition, exo activity stops, and APendo activity
remains and will break the APsites to give RF II. Another is to use beta
elimination by pH 11.5 buffer for 90 minutes at 37 C to break the DNA at
the AP sites. While the CsCl step should not produce AP sites or DNA nicks
if precautions are taken, it certainly can if the DNA is exposed to a good
amount of light. The two places that happens is if you used UV fluorescence
to see the DNA band in the CsCl gradient, or if you left the DNA in a trace
of EtBr in room light after attempts to remove most of the EtBr. If you did
not expose the DNA to light in presence of EtBr, and if the DNA was always
handled in sufficient TE buffer, your DNA should not be at fault.
Another idea is to make sure you do not try heat inactivation to kill the
S1 nuclease. You probably know that such attempt will truncate the DNA at
AT rich regions to give multiple band-like products.
Hope this helps.
Tony
At 11:56 AM 7/13/98 -0700, Sandy Kielland wrote:
>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)
>
>
>
************************************
Dr. Anthony T. Yeung, Ph.D.
Director, Fannie E. Rippel Biotechnology Facility
Member, Institute for Cancer Research
Fox Chase Cancer Center
7701 Burholme Ave. Philadelphia, PA 19111
Voice: 215-728-2488
FAX: 215-728-3647
email: AT_Yeung@FCCC.edu
http://www.fccc.edu/research/labs/yeung/
************************************