From: [Only registered users see links. ] [mailto:[Only registered users see links. ].indiana.edu] On Behalf Of peter
Sent: Thursday, June 21, 2007 12:43 PM
To: [Only registered users see links. ]
Subject: Re: Ligation
On Jun 20, 7:21 pm, "Deitiker, Philip R" <[Only registered users see links. ].edu> wrote:
Let me rephrase some of this
But I have had no success cloning 6 @ Bgl-II -Insert -- EcoR1
constructs into this vector. Even following protocols suggesting
10 fm of vector and 30 fm of Insert, all I get back are reclosed
vectors. *Bgl-II has compatible sticky ends with BamH1
Originally the situation was very bad, many vector colonies, but I
worked on the digestion to reduce the number of colonies to about
1/10th, approximately 10 vector closures per 10fm of DNA.
The other issue is transformation. Many here say use 1 ul of
[Ligation mixture]DNA for transformation.
So my thinking here is that if 1 ul is adequate my ligation is really tanking.
The only transformations are the [Insertless] vector, somehow unclosed after hours
of digestion, near complete digestion in 1 hour with either enzyme
(when tested independently and CIP for several hours.
The vector is grown, Minipreped, restriction digested (monitoring
timepoints by gel) CIP, promega SV PCR cleanuped, prior to reaction.
As for the Insert.
After restriction digest I cleaned the DNA up with "Gene Clean Turbo",
more recently because the mid range ligation is 30 fm I did a EtOH
precipitation completely dried the DNA and resuspended with 1/10X
warm ligation buffer. So there should this is now three purifications
removed from the last Gel. Still no success.
Is it worth making new primers and starting from scratch? is it a
common [Problem]primer that a certain sequence of overhand works
and a very similar overhand sequence does not?
~If you want advice/troubleshooting you would have first to tell us about the controls you ~have made:
~1. Vector digested in lig. Buffer
Why would I digest the vector in ligation buffer?
All transformed cells were grown, minipreped and purified
DNA was cut with Either BamH1, or EcoR1 + HindIII.
Vector transformant cfus were not grown, because
essentially the same number of transformants with insert
were all essentially vector.
2. Vector digested in Lig. buffer + ligase
The vector was ligated alone with ligation buffer + ligase
The number of colonies was exactly the same as with
insert + vector. 10 colony forming units/6 ul of Ligation
reaction (12 ul total). With the more rigorous digestion
and CIP this is down from 60 to 80 cfu per 8ul previously
obtained. With one ul there were approximately 7 vector
colonies per vector+insert reaction. 2 of each were tested
by restriction digest on 6 different inserts. All were vector
none had insert. A previous experiment with vector alone at 1/3 the
concentration resulted in 4 cfu per 6 ul, indicating that
with additional digestion times and CIP times the vector reclosures
were reduced to by one magnitude.
Why would I digest vector in the presense of ligase?
~On a first look it seems that you have too much CIP for too long, 1/10 of ul, in regular
~restriction buffer for not more than 15 min at RT will be enough, purify from gel after CIP
You mean buffer 2 or 3. I was told not to purify restriction
digests on gel before ligation as this interferes with trans-
formation efficiency. If the fragment can be cleaned off
on a column (less than 20 nt) that is what people recommend?
OK, but not sure why the vector can reclose on itself if the
ends are chewed off. All ligation products recut with both
BamH1 and EcoR1. Infact, that is how I know the ligation failed
because BamH1 cuts the circular DNA into linear DNA. But to
confirm I also tried to cut out insert and there was no insert
fragment. The folks from Promega assured me that the cloning-
insert in the vector, 12 nt in length, should be qualitatively
removed during purification on their SV pcr and gel cleanup
column, and it doesn't seem like it would be stable enough to religate
anyway. So the only thing I am thinking is that
somehow uncut vector was preserved through the digestion and
purification, even after 100 fold overdigestion, not obvious
on the gel during quantitation.
~3. Vector digested in Lig buffer + ligase + kinase
Kinase? There are about 200 varieties of Kinases?
Is there a protocol that calls for kinase?
Based on two replies it seems that the opinion is that CIP should be a problem.
So I am going to focus on this and run some controlled experiments.