(by peter.ianakiev from gmail.com)
Thu Jun 21 20:25:47 EST 2007
On Jun 21, 5:12 pm, "Deitiker, Philip R" <pdei... from bcm.tmc.edu> wrote:
> -----Original Message-----
> From: methods-boun... from oat.bio.indiana.edu [mailto:methods-boun... from oat.bio.indiana.edu] On Behalf Of peter
> Sent: Thursday, June 21, 2007 12:43 PM
> To: meth... from magpie.bio.indiana.edu
> Subject: Re: Ligation
> On Jun 20, 7:21 pm, "Deitiker, Philip R" <pdei... from bcm.tmc.edu> wrote:
> > I caught this quote in a message but I don't have the author.
> > "
> > Almost all ligation failures are really failures in preparation of the
> > DNA, not in ligation, and not in calculating or varying vector:insert
> > ratios. The ligation reaction is remarkably forgiving, especially for
> > cohesive ends. If you are having trouble, look at the restriction
> > digests, the treatment of the DNA following restriction, the
> > purification of DNA following PCR, the overhangs of the cut site on
> > PCR primer addtion of cut sites, UV damage to DNA during gel excision,
> > and only rarely at the ligation itself.
> > "
> > I am trying to clone several fragments into a derivative of E4276 a
> > derivative that has a BamH1 - - - - EcoR1 cloning site.
> > I have had successful ligations in the past, I managed to make several
> > clones of the nascent vector and they cut true to the restriction
> > sites.
> 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?
> > Thanks in advance.
> ~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.
If you don't understand what is going on, better do all the controls I
mentioned in the previous post.
1. Digested and purified Vector + ligase buffer ( this is your
background of un-digested vector)
2. Digested and purified vector + ligase buffer + ligase ( this will
give you how efficient CIP worked)
3. Digested vector + ligase buffer + ligase + T4 PNK ( this will give
you the amount of intact ends in case you have single digest (or a
partial one - when only one enzyme worked) . If you have single enzyme
cloning this control should give you lots of colonies. If you have two
enzymes this control should not give you lots of colonies.
4. Control vector (for example 1pg pUC) to estimate how good is you
cloning efficiency (cells may go bad, technique etc.)
5. Insert alone - if you make this insert from PCR you might have some
carry over plasmid template
6. Cells alone without any vector - to determine how efficient is your
Abt in killing this bacteria
1,2,3 should have same amount of digested vector, if you can do
sequential digest with the two enzymes even when the work in the same
Hope this covers most of the bases,
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