CHEF gel genomic DNA restriction

Hi

I have lately tried to restrict the genomic DNA of mammalian cells in
agarose plugs and then separate the fragments with CHEF pulse field.
The restriction seems to be working but the restricted DNA migrates
weird in gel. It is very dense in the Kb-Mb range (EtBr staining shows
that the DNA in that range has kind of spread over the lane borders).
After the dense part ends the DNA occupies only the central part of
the lane. Southern plot of the gel shows that the DNA runs higher
(more slowly) than it should be, also the bands are a bit distorted.

I was wondering if the anomaly might be associated with the
concentration of DNA in agarose plug, to my calculations there should
be about 3ug of genomic DNA in one plug. If that is the case then
could I vary the pulse field parameters so that the high amount of
genomic DNA would migrate normally?


Kaur Jaanson

Too much salt?

Wo

Hi

Sorry, forgot to add the conditions.

I use 1.2% agarose gel with 0.5x TAE buffer, running parameters are
5.4 V/cm field strength, 2-90 sec switch time, 19 hr run time.
Biorad CHEF DR-II system to run the pulse field electrophoresis.

Kaur Jaanson

On Feb 11, 2008 11:23 AM, Prof. Piero Sestili <[Only registered users see links. ]> wrote:



--
Kaur Jaanson

Hey,
i found this one on net.
check if it can troubleshoot some of your problems.
I think looking again into your protocol would do lot of help.

[Only registered users see links. ]

Sudheendra.

On Feb 11, 2008 3:05 PM, Kaur Jaanson <[Only registered users see links. ]> wrote:




--
Think before agree
Think before you nod
but STOP thinking
and You Are God

Hi

I have already read that manual, unfortunately it doesn't say anything
about the DNA concentrations effect on the mobility.
I am currently trying to test if the restriction enzyme buffer might
cause the problem.

On Feb 12, 2008 6:43 AM, Sudheendra Rao N R <[Only registered users see links. ]> wrote:



--
Kaur Jaanson