(ng of vector X kb size of insert / kb size of vector) X (molart ration of (insert/vector).

I always take the vector amount 50 ng and adjust the insert amount according to the above equation. Molar ratios which were most of the time successful for me was 1:1; 1:3 and 1:5. Also, the ligation time is important. Usually I use either 1 h ,22 ̊C or 18 h , 16̊C.

Also, if you try to increase the amount of your digested vector and insert (by increasing the reaction volume) that might be a good help.

Good luck

Yep. For double sticky end you need molar ratio to be 3:1 insert:vector. So you need to know the vector size. This you can see on the gel you had from the double digestion. If calculations is not your thing there are plenty of calculators online to do the math for you. Look at the websites of re enzyme vendors. If the ligase you use has an concentration of 3U/ul use more of it. You can't over ligate. Do not exceed 100ng DNA total in a 50 ul dh5 transformation. The cells don't like it. Also don't add more then 10% of the transformation volume. I usually do ligations in 10ul and do not exceed 200 ng. I add 1 ul of ligase 3U/ul. Ligase is indeed ATP dependent. So use fresh ligation buffer. Best to freeze small aliquots. ATP breaks on freeze thawing. Ligase is a lot more stable and efficient then most people think. But for difficult ligations ( which yours isn't) you could try o/n at 16C.

If these suggestions don't work your problem is probably elsewhere. Ethanol in DNA prep for example.

Good luck!

Usually we use 1:3 insert : vector for this type of ligation....use the below calculator which will be help full to u...

http://django.gibthon.org/tools/ligcalc/

Hi, as suggested by other scientists follows 1:3 ratio with size calculation at proper concentration, Take vector 50 ng..

Few points:

1. Incubate for longer time than racoomnded

2. You can use PEG, even for sticky end ligation

3. Always have a positive ligation control, you can use DNA maker (eg. lamda DNA HindII digested).

4. Before the transformation you can do a PCR from putative ligated product by using one vector and another gene specific primer and look for expected size on Agarose gel.

Thanks all for their useful comments.