Ligation

[peter]

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.
>
> 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.
>
> 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 ends of the Insert contain
> C-A-C-G-G-A-G-A-T-C-T-T-G-G - - - - -
>
> According to NEB this should be enough overhang.
> as this
> . . .  T-C-C-A G-A-T-C-T-T-C-C - - - - -  cut to 100%
>
> The EcoR1 site is the same thing
>    C-G-G-G-A-A-T-T-C-A - - - - - - - - - - (My insert)
>
> t-c-g-a-G-G-A-A-T-T-C-C - - - - - - - - (NEB, lower case = single stranded overhang.)
>
> The other issue is transformation. Many here say use 1 ul of DNA for transformation.
>
> I don't know if this works or not, but I have linear increase in clones
> from 1,2,4,8 microliters. More than 8 uls of ligation mixture did not result
> in more clones and eventually clones declined.
>
> So my thinking here is that if 1 ul is adequate my ligation is really tanking.
> The only transformations are the 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 clean 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 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
2. Vector digested in Lig. buffer + ligase
3. Vector digested in Lig buffer + ligase + kinase
4. Positive control vector (for transformation efficiency)
5. Insert alone

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

my2c
Peter