RFD: Plasmid re-ligation without ligase?

Matthew L. Brown, Ph.D gemini3 at mindspring.com
Wed Apr 23 15:11:12 EST 1997


Wolfgang, Klaus, and Rafael,
	I thought I might get into the fray a bit. First, Wolfgang, I have
observed very similar results before but never was so clever to
attribute the result to the E. coli being able to ligate or read over
the unligated plasmid ends as suggested I believe by Klaus. I on the
other hand attributed the odd results to a minute amount of
contaminating uncut plasmid as mentioned I believe by Rafael. I have
also had other experiences that suggest that the contamination theory is
plausible. For example; I routinely purify digested bands onto NA45
paper or purify directly from the agarose. On occasion I have either
gotten strange transformants. Upon sequencing or PCRing to confirm
insert I have seen that the plasmid seems to be the mother (uncut)
plasmid or another plasmid entirely (now that was really confusing or
maybe not). Let me explain, I use but two electrophoresis apparatuses
and usually switch between gels with little more than a deionized water
rinse between usages (I know, not a great technique). I also use pGEX,
pBluescript, and pCRII (TA cloning vector) frequently nearly
simultaneously. [i.e often I am cloning PCR products (pCRII),
restriction products (pBluescript), and making fusion proteins (pGEX) on
or about the same day] (busy little Post Doc) Anyway, I have by
sequencing confirmed the presence of incorrect plasmids both with and
without inserts in some cloning reactions. Now I will try to explain my
theory on how this happens. When using a a "dirty" apparatus used for
different kinds of plasmids or when running uncut vector or vector plus
insert on the same gel as cut material it is possible (Rafael?) for two
or more types of DNA to comigrate in the gel.(?) Also when purifying on
NA45 paper (high affinity for any nucleic acids, even contaminant)or
from gel directly (residual running buffer or gel may contain the
contaminant) a small amount of a contaminating plasmid may persist. I
often concentrate my insert DNA and use most of it in a ligation
reaction with a dephosphorylated vector to help insure effective
ligation of insert into vector. If a contaminant carries over with the
insert DNA then it is in the ligation reaction. When transforming cells
the successfully ligated inserts plus vector are still nicked (due to
dephosphorylation of vector) and not supercoiled. This reduces their
efficiency to transform.(?) Ah, but here's the rub, if a contaminant is
present even at a low level and is supercoiled it will transform more
efficiently.(?) Therefore, with a weak or failed ligation reaction and
transformation the only clones present may be the result of this
contaminant. Moral of this story, try to use clean equipment, don't work
with multiple vectors on the same day, avoid purifying inserts on gels
that also contain uncut vector or vector plus insert, and be wary of a
weak or mildly successful ligation/transformation because it may yield
only contaminant containing clone. This is only my theory. By the way,
back to Klaus' suggestion about the E. coli helping out in the
religation. I don't want to dismiss this as a possibility. One thing
I've learned with working on microorganisms is that when you think that
you have figured them out, they go and change the rules!
Sorry for my rambling.

Best of luck,

Matt Brown, Ph.D.
Research Fellow
Dept. of Medicine
Univ. of Alabama at Birmingham



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