Annie Chamberland wrote:
>> hi!
>> i read a article about PFGE with xanthomonas being digest with the XbaI
> enzyme and they got lot of bands.
> i dont have the equipement for PFGE so i try to digest DNA from 10 samples
> of Xanthomonas with XbaI for 20 hours like the PFGE protocol and made a
> normal agarose gel but i didnt get any band. I also try DraI but got the
> same results.
> It work with other enzymes like BamHI and EcoRI but i got too many bands.
> I want to know if its normal that my DNA wasnt digest with the enzyme XbaI ?
> Does the PFGE have an effect on the digestion of DNA?
> Hope someone can help!!!!
> Thanks a lot!
Isn't XbaI from Xanthomonas badrii? That could be your biggest problem
as this one is blocked by methylation (I got that from the NEB
catalogue)...
Well, if you're looking for ANY XbaI cuts, then here's my 2 cents,
PFGE is used to electrophorese very large (100's of kilobases to
megabase-sized) fragments of DNA. The one we use have a hexagonal
arrangement (called CHEF, for contoured hexagonal electrophoretic field)
for moving the DNA fragments through the gel by yanking them back and
forth. Using this specialized form of electrophoresis became necessary
not only for mapping studies, but because very large DNA fragments will
not resolve on an agarose gel, even at lower percentage concentrations.
On a regular (0.3-1.0%) gel, the bands tend to congregate at around 23
kbp. Even when running at slow speeds overnight, one can't resolve over
30-40 kbp. If your enzyme cuts only a few times, then it is very likely
that you will never see the bands on a regular gel.
PFGE equipment is extremely expensive. This is in part because, in
order to run one, you will need the electrophoretic chamber, a pump to
recycle cold buffer, a power supply that is able to "ramp" up or down
the voltages needed to run the gels, equipment to set up the gels, and
equipment to make agarose plugs for running on gels after digesting (and
I am sure I forgot to list a lot of stuff here). DNA has to be prepped
in agarose plugs to prevent shearing; this includes lysing cells,
removing other cellular components, and digesting the DNA after
isolation.
You could try to end-clone the pieces by cutting with a separate enzyme
(do some experimentation here) and directionally cloning into a vector
(one cut with Xba and the other enzyme). The # of different inserts
could be checked via comparing restriction patterns or probing on
Southern Blots. Sounds messy but should work, theoretically.
If anyone knows more, please correct me if I am wrong on any of the
above or if I left anything out. I have been doing this (pulse field)
for only a short period of time.
BTW, is there any particular reason to use XbaI?
Anyone have any additional thoughts?
--
C. J. Fields
Graduate Student, Dept. of Biological Sciences
The University of North Texas
Denton, TX
email : cjfields at jove.acs.unt.edu
