Blunt end ligation - (Jun/26/2008 )

Hi

I am doing blunt end cloning of the two DNA fragments of insert size 3.8 kb and vector size of 5.6 kb. It is a Hpa1 site. After ligation i am not getting colonies. I run the ligation product and i find that the liation product looks like a ladder on the agarose gel. Can anybody suggest to prevent this type of ligation and get the colonies succefully.


sunil

hi

let us know your ratio of insert / vector

cheers,
akhshik.

I have no good experiences with blunt end cloning, be sure that ratio insert vector is good, 1:3 or 1:4 because recircularization is preferentialy done. Let ligation run overnight at 4 degree of Celsius in 30 ul and before transformation run it in the gel to see efficiency.
or clon it by kit and subclon latter by PCR
good luck

Blunt end ligations are less efficient in general than sticky end ligations, and I suggest that you have a 3-5 to 1 ratio of insert to plasmid when ligating. (you may sometimes use ratio like 8:1) ligation at 4 degrees, is better than 14 degrees overnight.

cheers,
Masoud.

I usually do the ligation at 16C and the ratio insert:vector is 1:4

You got no colonies so firstly make sure the competent cells are ok. If so, then there is no ligation occurring at all. This can be a good thing because at least the vector is not producing background colonies. Because the ligation is not occurring, you need to use more of both the vector and insert to get the ligation happening. I'm not sure exactly what your conditions are, but for this difficult ligation use a good amount of vector (100 ng or more) and make up the rest of your reaction with your insert (no water). The large amount of vector is fine because it is not producing background colonies and the large amount of insert will force the insert into the vector and create the molar excess of insert you like in a ligation. More units of ligase will also help the ligation. High concentrated ligases are useful in this situation because they add more ligase to the reaction without stealing volume. In terms of incubation temperature, i usually do 1 hr @ room temp and transform that and then incubate the same tube O/N @ 16C so that if the first transformation does no good i can transform the ligation after the longer incubation period. Sometimes the ligation is done after an hour but other times the O/N works well. If all this fails, make another vector prep or prehaps try a different strategy - sticky end ligations are much simpler.

I would also make sure the ligase is working, by treating some DNA ladder with ligase, and then running a gel. Your ladder should lose the lower bands.

150 ng of a 3800 bp fragment = 60.7 fmoles or 7.3e10 ends.
221 ng of a 5600 bp fragment = 60.7 fmoles or 7.3e10 ends.
Blunt end ligations work great with 60 fmoles of vector/20 uL ligation.
You want a 1:1 ratio of ends for the most efficient ligation.

Assuming that your cells are competent and you are getting colonies when you transform supercoiled DNA:
If you have not dephosphorylated the vector and are getting no colonies in a vector-only ligation, then the Hpa I ends are probably damaged by endonucleasses or the DNA has been harmed by too much exposure to UV during gel extraction.