More Bacterial Chromosomes and PFGE
lanoilb at ava.bcc.orst.edu
Tue Sep 6 13:08:37 EST 1994
This is a follow-up to a post I made before. I feel that a little more
detail in my protocols and what I observed might be helpful for people to
better understand my problem. Thank you to those who have already responded.
I have been using CHEF to separate and observe chromosomes from the
marine bacterium Alteromonas haloplanktis. As a control on these gels, I
have been running undigested whole E. coli chromosomes. I prepared the
plugs by treating the log phase cells with chloramphenicol (0.2 mg/mL) for
1 hr (approximately 3 doubling times) to allow completion of replication but
not reinitiation. I then sealed the cells in agarose plugs and digested
for 16 hours at 37 C with a lysis buffer containing lysozyme, EDTA,
and salts. Then I digested further with a solution of 0.5 M EDTA, 1%
Sarkosyl, and 1 mg/mL proteinase K for 24 hours. I then washed
extensively with 0.5 M EDTA.
Using a switching protocol optimized to separate in the range of ~350 kb
to 5 Mb, I then separated the chromosomes out without any linearization
or digestion. I observed multiple bands in the E. coli lane, the
smallest of which was the predicted size. A similar pattern was seen in
A. haloplanktis, although the size of this genome is not known. The E.
coli strain contains no plasmids, and is, to my knowledge, a complete
chromosome. Since large circular chromosomes do not enter the gel during
PFGE (?) unless fortuitously nicked during preparation (in which case
they should run at the correct size for the linear version), I am asking
what could be causing these extra bands.
Several people have responded that the extra bands are concatemers of the
chromosome which are formed due to the inability of the chloramphenicol
to affect the cells fully, either due to concentration or time. I have
tried several different concentrations, and I have seen no effect,
although the lowest that I have gone is 0.1 mg/mL (it is difficult to do
these experiments since these gels take 5 days to run). I have not
attempted different times, but I think that it is unlikely that this is a
factor since one hour is approximately 3 doubling times for E. coli and
almost 2 doubling times for A. haloplanktis.
I appreciate the response I have gotten already, and I thank you in
advance for any advice you might give.
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