From: [Only registered users see links. ] [mailto:[Only registered users see links. ].indiana.edu] On Behalf Of ChenHA
Sent: Thursday, June 21, 2007 2:19 PM
To: [Only registered users see links. ]
Subject: Re: Ligation
Deitiker, Philip R wrote:
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.
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 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.
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
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.