Ligation

[Deitiker, Philip R]

-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of ChenHA
Sent: Thursday, June 21, 2007 2:19 PM
To: methods from magpie.bio.indiana.edu
Subject: Re: Ligation

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?
"

Bacterial cells? The construct is in a mammalian CMV promotor
with excellular leader sequence. I am trying to clone 4 different
domains, only one of which is enzymatic and 3 appear to be regulatory. 
So far as yet I have not been able to get full length or domains. 

The protein I am cloning actually has the ability to target itself
anywhere inside or outside the cell. Which is why I am trying to
pipe it outside the cells. It can potentially kill a cell. 

> 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.
"

I was thinking the same thing. But, my insert has 5 BamH1 sites, that is the reason I BglIIed
them. One of the domains does not have a BamH1 site so I can try this technique. My thinking
is that the religation is not the real problem, since the number per unit DNA and given the amount transformed, its very low. You talk about 100ng of Vector. 6ul at 280 ng per microliter
and given 6 ul of ligation comparing that to 100ng with 1 ul of ligation I should expect no vector-only at all. But how is the vector surviving with its cloning sites intact. This is
confusing me. If I got a bunch of blunt end ligations with cloning site gone then I could
positively say the CIP was the cause. 

"
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.
"

OK, this I like. No CIP. I am going to try a test ligation with HindIII, EcoRI, and/Or BamH1 and no CIP. HindIII and EcoR1 can be heatkilled. I'll see how it works.

> 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.
"

This is about right 6 ul per 50ul of DH5[alpha] competent cells. 
  
> 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.
"

I did a NaAcO/EtOH precipitation. In order to avoid any possibility of EtOH in the ligation
I heated the DNA to 65'C until it all EtOH was removed. The DNA was resuspended in water.
The resuspended DNA remained stuck to the microfuge tube so I heard that adding warm water
was required and a little salt helps bring DNA back into solution. Rather than add another variable I used a hyposaline amount of ligase buffer and heated it to 37'C and the DNA came right back into solution. 

"
If you are preparing a vector DNA fresh, always do a test ligation with no insert - this is to check the background religation.
"

Yes, I have been doing this. I am going to make up new vector this evening, I will
icksnae the CIP and check the efficiency. If it is high I will try doing maybe
a 30 minute CIP, at the end of the digestion, gel purify, reCIP 30 minute and
then hit it on the SV PCR column as a final cleanup. That should take care of
any contaminants. 

"
  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).
"

This is not a problem, I have been doing this anyway. 

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

I have no idea what the efficiency is, but these are,I believe,Invitrogen DH5alpha. Aliquoted
and stored at -80'C. I inherited them from a previous coworker. I had about 2 weeks to learn all the cloning stuff again so . . . . . [Protein chemist babbles on :^)] I routinely transfect these cells with intact vector, I get 70 or 80 colonies with 1ul of DNA. 

OK, guys, thanks for the advice, now I need to do some work and I will get back
after I identify the problem.