pCAT Basic cut by Hind III

cjharrod at waikato.ac.nz cjharrod at waikato.ac.nz
Sat Jun 25 04:42:05 EST 1994


In article <CrKC86.3FD at acsu.buffalo.edu>, vp23qw83 at ubvms.cc.buffalo.edu 
(Xin Guo) writes:
> Hi, bionetters:
> 
> I cut pCAT Basic (~4.4kb) using Hind III (one Hind III site) and
> got three bands on agarose gel. Compared with lamda DNA/Hind III
> marker, I saw one the 4.4kb cut linear pCAT band and non-cut
> circular pCAT band. But I also saw a band about 8.8kb. The three
> bands seem to have the same amount of DNA.
 
> Xin Guo

If you see three bands after a restriction digest, I suggest that your 
restriction digest was incomplete, and you are seeing Open Circular DNA (the 
"8.8 kb" band), Linear DNA and Closed Covalent Circular DNA.  (See Sambrook et 
al.  Molecular Cloning 2nd Edn. CSH Lab. Press 1989, page 6.5 for more detail).

Causes for incomplete digestion include:
  
	i.   Insufficient restriction enzyme used.
	ii.  Restriction enzyme is too old.
	iii. DNA is not clean.

The third option is most likely, particularly if you prepared the DNA using
the Phenol:Chloroform method without further purification.  DNA preparations
which are not clean (i.e. have protein bound to the DNA) retain DNA in the
wells of the gel after the gel has been run.  If you see this, your DNA is not
clean!

I recommend that your further purify your vector using a Cesium Chloride 
gradient, as this will remove protein, chromosomal DNA and nicked (Open
Circular) DNA from your preparation.  (See Sambrook et al. Pages 1.42 - 1.45).

Remember one of the central caveats of molecular biology:  

	The cleaner your DNA the better it will work!

Hope this helps!

Chris



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