Subcloning questions

Timothy Paustian paustian at facstaff.wisc.edu
Thu Mar 2 13:39:20 EST 1995


In article <3j4oj9$p5p$2 at mhadf.production.compuserve.com>, David J. Kanter
<75444.2004 at CompuServe.COM> wrote:

> Got some questions on a DNA subclone/restriction map lab I did.
> 1. In one well of my gel separation, I saw a bright haze in the 
> bottom lane rather than a band. Is the sample degrading as a goes 
> across the gel? What’s going on? (Used ethidium bromide with E. 
> coli vector)

My guess is the smear is RNA. Contaminating RNA will come through many
plasmid preps unless RNase is added to get rid of it. Or you may be right,
your DNA is getting chewed up by a nuclease. You can test this by
incubating a sample at 37°C and one at 0°C for the same amount of time and
then running. If the 0°C has a plasmid band and the 37°Ç one doesn't,
nucleases are probably acting. 

Another possibility is the lane contained chromosomal DNA and when you
digested with the rest. enz. it cut it into many differently sized pieces.


> 2. Why ultracentrifuge to purify DNA?
CsCl density gradients are an effective (if a little outdated) way of
getting very clean DNA. The Ethidium bromide that's added will intercalate
in both plasmid DNA and chromosomal DNA. However, since undamaged plasmid
DNA is supercoiled, it cannot bind as much EthBr/bp and has a different
density than chromosomal DNA. In the CsCl gradient, chromosomal DNA
seperates from plasmid DNA due to this difference in density.

Recently kits have been manufactured (I use Magic preps from Promega).
These kit eliminate the need for CsCl gradients in many cases--providing
DNA that is very pure. 

> 3. Why would a restriction enzyme NOT cut a plasmid? Because a 
> protein in gel bound to restriction site? Why partial digests?

In many cases the enzyme or DNA was altered by something in the mix. Here
are a few possibilities.

1) Proteases in the purified DNA that kill the restriction enzyme

2) The wrong restriction buffer was used and the enzyme as suboptimal activity.

3) Nucleases, although this would give a smear of DNA, not the partial
digests you report

4) The DNA is methylated and the restriction enzyme cannot cut it.

5) Old restriction enzyme.

there are many other things that could go wrong that I probably forgot to
mention.

Hope this helps.

Tim

-- 
Tim Paustian                   "Life is a journey, not a destination"         
Department of Bacteriology     
University of Wisconsin-Madison
1550 Linden Dr.
Madison, WI 53706      Email:   paustian at facstaff.wisc.edu



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