Ligation

[ChenHA]

Deitiker, Philip R wrote:

> 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. 
>  

Do you suspect that the protein may be toxic to the cells?


> 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.) 
>  
>  

Just an idea - since you are ligation BglII to BamHI, then what you can 
do is add BamHI to the ligation mixture.  The religated plasmid will be 
digested, but not the one with insert.  (This is a useful trick in blunt 
end ligation, but may be applicable in your case).  Something to think 
about, but for directional cloning you really don't need to do this.

You actually don't need to do CIP treatment to start with because you 
have two different ends, so using CIP is rather pointless, just causing 
more problem.  In my experience CIP should be avoided unless absolutely 
necessary.


> 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. 

The standard advice is to use a volume equal to 5% of the competent 
cell, i.e. if you use 100 ul of cell, then use 5 ul of the ligation 
mixture.  This is to avoid having too much contaminants which may affect 
the transformation efficiency.  However, I regularly use 10 ul of 
ligation mixture in 100 ul of cells with no problem, and as you found, 
you can use more without problem.

>  
> 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?

Normally I just add 7-8 extra bases to save me the bother of checking. 
Here's what I do with ligation -

Digest, and then I always gel purify my digested DNA, and always use low 
melting point agarose (this may be unnecessary, but since my ligations 
nearly always work, there is no reason to change the way I do it, even 
if just passing it through a column is faster and should give reasonably 
clean DNA).  Don't bother with EtOH precipitation.

For ligation, use 10-100 ng of vector DNA, and 1-3X insert in 20 ul of 
reaction.  Heat DNA mixture at ~50 degC for 5 minutes, cool, then add 
ligase and buffer.  Leave ligation O/N in fridge (why do you use warm 
ligation buffer?) then transform.

If you are preparing a vector DNA fresh, always do a test ligation with 
no insert - this is to check the background religation.  Often I get 
5-20 X more colonies for those with insert compared to those without, 
and  you can then be sure that most of the transformants has the plasmid 
with insert.  Even if you have an equal number of transformants compared 
to the test ligation without insert, a good proportion will have the 
insert, just need to do an extra screening step (say, by PCR).

The most important thing is having good competent cells.  Transformation 
efficiency of over 10^8 is ideal.  And no CIP.

Lastly, someone posted something about cloning methods without ligation, 
you can try those if this continues to be a problem.

> Thanks in advance. 
>  
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