Basic Subcloning Question
chrisb at hgu.mrc.ac.uk
Tue Jun 1 10:14:40 EST 1999
Orac (Orac_USA at hotmail.com) wrote:
: In article <7ikjck$j2u$1 at news.usf.edu>, "Sean Yoder"
: <syoder at chuma.cas.usf.edu> wrote:
: >I'm still green in molecular biology so please bear with me. My question is
: >regarding my first attempt at subcloning. I'm inserting a large 3.1kb EcoRI
: >flanked insert into a vector (4 kb) cut with the same enzyme....the problem
: >is I did not dephosphorylate the ends of my vector before ligating...Doh!
: >I'm going to assume that tomorrow I will have thousands of transformants on
: >my plates.
: > Should I just start again and dephosphorylate this time or do you think
: >I have a half decent chance of finding a transformant with the insert...I
: >can screen dozens at a time for protein expression with an immunodot which
: >will indicate when indicate the presence of the insert as well as correct
: >orientation instead of running dozens of minipreps. FYI the vector is a
: >pCMV euk expression vector and the only marker is Kanamycin resistance to
: >those with the plasmid.
: > It's gonna be another long weekend in the lab...
: I'd *strongly* recommend dephosphorylating your vector and redoing the
: ligation. While it's true that you only need one good clone, finding that
: clone would be the hard part if you forgot to dephosphorylate the vector.
: Thermodynamically, reclosure of the vector is favored so much over
: reaction with your insert that, unless you get incredibly lucky, you'll
: have to screen hundreds (more likely many thousands) of colonies to find
: one with your insert. Worse, you could screen every colony you have and
: still end up with nothing but religated vector. That's a lot of work for a
: low probability of salvaging the experiment.
Where did you get that idea? It's simply not true at all. I have done
scores of sticky-end ligations without phosphatasing the vector and get
positive clones at a frequency of up to 70% on occasion, with the range
usually being from 5-50%. Reclosure of vector can be minimized to < 50%
if (a) fragment size is as small or smaller than the vector, (b) the
vector:fragment ratio is optimal and (c) the concentration of ends is
sufficiently high. The maths demonstrating this is complicated
(although if you think about it you will see it must be true), but the
empirical evidence is overwhelming: phosphatasing did not come into
common practice until many years after cloning started but people still
managed to come up with the goods.
(Of course if the ends are blunt, all bets are off and it's much better
-- though not essential -- to phosphatase vector.)
: It's *far* easier and faster just to redo the ligation the right way.
: You'd only lose a couple of days and you'd have a MUCH higher probability
: of success. Better yet, you wouldn't be stuck long hours over the weekend
: screening colonies with a low probability of finding a good one.
No, it would have been well worth screening the colonies, preferably by
Chris Boyd | from (but not \ MRC Human Genetics Unit
Christopher.Boyd at hgu.mrc.ac.uk | on behalf of) / Crewe Rd, Edinburgh
http://www.hgu.mrc.ac.uk/Users/Christopher.Boyd EH4 2XU, SCOTLAND
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