Has my ligation work? - (Jul/07/2005 )
What should properly ligated product look like when you run it out on a gel?
I know you are not supposed to see your vector band or your insert band but what is a good indication that ligation has work?
When I ran my ligation product out I got two smears, one above 10kb, and another from 6kb to 2.5kb. Is this good or bad? My vector is 5.5kb and insert is 3.2kb
HI,
The correct product forms a circular DNA, so it
should look smaller than the actual MW and
appear as several bands.
So it is a good sign that you saw bands smaller
than 8.7 kb, which is the size of the product.
However, formation of a band of 10 kb may not
be a good sign because it indicates incorrect
products such as concatemers.
I'm not sure about what other people think, but
from my experience a lot of large DNA is a bad
sign.
Hi, I'm a newcomer to this bioforum and also a baby in this field. Just a bit curious about the question you asked.
I was wondering, couldn't you just digest it with RE and run gel? Coz you know your vector size and your gene of interest's size. If your ligation works, it'll definitely be there I think?? And, I thought you could also transform it into bacteria and plate it? And then you could see from the white colonies? 
True, only if you cloned it into a vector that has a c-term laqZ sequence and you plated on X-gal containing plates.
To see if a ligation may have worked you need to run the negative control (cut plasmid+ligase and no insert) along with insert ligation on a gel and see if there are any novel bands of approximatly the correct size in the insert ligation that is not in the control ligation.
Other then that, transform both ligations (separatly) into the competent cells and spread on plates. Hopefully your negative control will have much fewer colonies then your test ligation.
*Which reminds me, I have a transformation in the incubator* Lets look and see what happened....Negative control 4 colonies, the two ligations with insert... in excess of 250 colonies. I would call that a successful transformation.
Well, I'm not exactly seeing any bands of appropriate size when I ran out my ligation product, just some smears. According to my boss my ligation has worked. But I haven't been able to get any colonies at all. I tested the efficiency of my competent cell and found that it's around 3x10(exp 6). My boss thinks that it's a competent cells problem i.e. it's no competent enough.
So anyway, I'm in the process of preparing more competent cells to see whether I'll better luck.
haha, someone said that ligation is an easy work in molecular biology, but it definitely troubles us.
Consider your result on the gel, i think maybe there are some contaminates or some damages to your DNAs. You'd better run the fragments and ligation solution on one gel, and take them as control.
In my mind, the ligated product is not so much that you can clearly see on the gel. And i think if there is a smear around the currect size is also OK.
It is just my opinion.
Good luck.
Hi Muly,
I'm new to the bioforum.
Since your vector+insert size is ~9kb, I think its better you transform electrocompetent cells by electroporation where the efficiency is higher.
All the best
Reena
Hi,
I know you are not supposed to see your vector band or your insert band but what is a good indication that ligation has work?
I had ligation problems as well and was told to run the following two reactions on a gel :
1) (a part of) my ligation reaction and
2) a reaction with insert+vector but without ligase.
What I saw was
1) a high molecular weight band of the approximate size of my ligation product (this is circular DNA which is supposed to run higher than the linearized or supercoiled product); I saw neither linear vector nor fragment left.
2) I saw the linear vector and fragment bands and absolutely no high molecular weight smear.
I assume this ligation had worked fine although I couldn't check this (unfortunately) because there was no ligation reaction left for the transformation (so you should always test only a part of your ligation reaction !).
In your case, your ligation might have worked in part as indicated by the high molecular weight smear (running a bit higher than 8.7 kbp).
The smaller smear probably indeed represents your insert and vector not used up in a ligation (why these bands don't run at the appropriate molecular weight I don't know.)
Why don't you try to run the reactions I described above ?
Hope this helps