band separation prob. on agarose gels

It all started when I wasn't getting any colonies on my plates, so I checked
the purity of my starting material...

I'm trying to do a simple sticky-sticky cloning step using EcoRI and NotI. I
cut 5 microg. of each plasmid. One plasmid yields 4.3kb and 2.5kb bands, and
the other plasmid yields 3.5kb and 1.6 kb bands. I digest both plasmids and
separate the bands on a 1% agarose gel in TAE at 5V/cm. I then soak the gel
in a TAE/EtBR solution and visualize. There is good separation of all bands
and digestion is 100%. I want to retrieve both larger fragments so I closely
crop them from the gel and extract the DNA using either the Qiagen gel
extraction kit or Qiaex II resin. I then ran a sample on a gel to assess the
purity and I was surprised to see contamination from the band I threw away,
and I'm not talking trace contaimination that one would expect: one of the
fragments is 50% contaminated with the wrong band and the other fragment is
30% the correct band, 70% the wrong band (judging by eye). Therefore the
fact that I get no colonies on my plates could be due to most of the
transformed material consisting of fragment-fragment and vector-vector
ligations.

On a lark I purified the correct bands from this double-separated sample and
used these for ligations: no colonies, but I did not run the gel a third
time to assess the purity or quantity of this material. I have been using
the same stock of 50X TAE since November, and it looks ok, I have been using
the same DNA load buffer for 6 months and 1 Kb ladder prepared using that
buffer today looks exactly like I expected. Both reagents provided success
in the past.

Has the physics of gel sieving stopped working for me? Any suggestions on
how I can fix my universe and recover only one fragment per band?

Kyle

Kyle Legate wrote:
<snip long description of purifying two fragments from one band...>

I received some good suggestions into how to fix this problem, but in the
end nothing worked so I threw out and remade all reagents except for the
restriction enzymes. That seems to have fixed the problem. Somehow it almost
always comes down to this solution, for specific reasons I'll never know.

Do you by any chance boil your DNA samples before running on a gel? Some
labs do boil DNA. If so, there is a chance that your particular fragments do
not anneal correctly, and rather form predominantly 2 species, one of them
running coincidentally with the band you want to get rid of. That might
indeed had to do with the reagents (loading buffer?) you used. Could that be
true? Otherwise, I am same flabbergasted as you are.
Emir

"Kyle Legate" <[Only registered users see links. ]> wrote in message
news:cdea459ff6bffb2bd990677c829976ab@news.teranew s.com...
almost

On Mon, 16 Feb 2004 18:14:31 GMT
"Kyle Legate" <[Only registered users see links. ]> wrote:


Did you inactivate the enzymes? How many wells did you used?

--
The propriety of some persons seems to consist in having improper
thoughts about their neighbours.
-- F.H. Bradley

Ferdinando Pucci wrote:
I used 2 wells joined together to accomodate that amount of DNA, and I've
never found a reason to inactivate my enzymes, since I never detect activity
after gel purification.

On Mon, 23 Feb 2004 18:03:47 GMT
"Kyle Legate" <[Only registered users see links. ]> wrote:


Only 2? Afaik you could have carry over problems, i suggest to try with
more wells (at least 5).


--
I am not an Economist. I am an honest man!
-- Paul McCracken