Hello, 

I have done some similar experiments few weeks ago, did you digest your insert from a PCR product? Or did you clone it in a first vector then digest it? What I do is: after PCR and purification by electrophoresis, clone the PCR product in a blunt vector, then digest with the enzymes needed and use the purify fragment as the insert. So you'll be sure the digestion has occured. 

Also I incubate my ligation at 16 celsius degrees overnight or at least 4hours. (those parameters depend on the ligase kit you're using)

Hope I've been of some help! 

I cloned it from the first vector and digested it. I believe your doing a TA cloning, correct me if I am wrong. My digestion works very perfect, I am having trouble with ligation I use NEB ligation calculator to calculate the insert to be ligated at 3:1 and 5:1 ratio I am still having trouble. Thank you for your response.

1.- Have you checked the integrity of the DNA fragments in a gel after the purification and prior to the ligation?

2.- Have you set digestions of the source plasmid (the one that already contains your insert) with either EcoRI or XhoI to check there is no unexpected sites that generates other fragments of around 500bp?

3.- Have you set a transformation efficiency transformation with non digested destination vector?

4.- Have you set a ligation control of the destination vector? Set two ligation reactions with vector digested with just one of the enzymes. Add ligase to one of the reactions and NOT ligase to the other. Comparing number of colonies obtained after transformation you will know how ligation is working.

5.- You can set a similar control using double digested destination vector. That will tell you how efficient the vector double digestion is.

6.- By the way, how big is the fragment that the destination vector releases when double digested? If it is just some bp, because two sites are too close, you can have problems because one of the enzymes is not efficiently cutting the vector (you can know this from the results of 5.- ). I would go for a sequential digestion. First with SalI and, after a couple of hours, adding EcoRI.

Good luck

Thank you Manuel Arroyo Mateos .

1) I have not run the DNA fragments in gel after purification coz the yield of the fragments obtained was to low, and I have been doing touch down pcr for my insert  and I believe that my fragments  obtained after the PCR reaction is pretty pure.

2)I haven't set digestion for source plasmid, I will have to look into it in future.

3) I have set transformation efficiency for empty vector and I have got only 20 colonies for 1ng. I made up new stock of competent cell when transforming empty vector there was more than 100 colonies. But when transforming my ligations no colonies were formed.

4)I have set a double digestion for vector and on transformation i see no colonies in that too.

5) When double digested, salI is at destination of 42 fragments away from EcoRI, i hope this should be alright and I add both the enzymes together and incubate them for  2 - 21/2 hours.