Ligation issues - (Apr/11/2009 )

Hi, I have been having problems with my cloning for the past 2 months. I was doing a three way ligation and it didnt work for a long time so taking a new approach with just a simple ligation and still I have been having issues. I cut with Sac I and Sal I create my insert and trying to ligate it to a vector that also has been digested with the same enzymes. Its a vector that has a Leu2 marker and ars1. and the vector is about 9KB while the insert is around 3KB. I have had problems gel puryfying my frgements as well since it seems that I'm loosing a lot of the sample.

I use the t4 ligase 0.5-1 ul and 1 ul buffer with 1 ul vector and 4-5 ul of insert, and add water to bring up to 10ul. My positive control (uncut vector) looks fine and I get lot of colonies but the rest of my ligations I have not been able to get a single colony. I tried 16C water bath over night and also I have tried to leave the ligation at RT for about and hr and then leaving in the water bath over night. and nothing has worked so far.
Any advise on anything I could change or do different will be a great help!.

thanks,

Hey,

Avoid running the vector on the gel. It is my personal experience that when I avoid agarose for large vectors, I get better ligation efficiencies. So perform the digestion, CIP the vector and precipitate. USe this for ligation with insert. Screen colonies and you should get a clone.

Best,
TC

T C on Apr 11 2009, 09:59 PM said:

Concentrate on the gel extraction . that's normally the part that messed ligation up if tat is ever involved.

Make sure to use as MUCH DNA as possible. FULLy load teh well . as concentrated as possible.

and be prepared for an etanol wash after gel extraction .

salt contamination is really a good friend with gel extraction .

beware of over exposure to UV light when gel extracting DNA bands. UV does harm ligation efficient particularly when working with long DNA fragments. Exposure time should be measure in seconds rather than minutes.

If possible set your machine to long wavelenght UV.

How much vector and insert are you actually using? Can you find out what is the concentration of your reagents? The ligation reaction is based on mol (number of molecules).

could you estimate the vector and insert conc by running on gel. how long do you digest the vector and insert? long digestions can damage ends and prevent ligation.