You may be transforming too much total DNA from the ligation. According to NEB, you should not transform more than 40ng total DNA into the competent cells. Since you set up different ratios for the ligation, you should be able to calculate the total DNA in the 5ul that you transformed. You may need to transform less volume or dilute the ligation to get under 40ng.

Also, how much of the transformation did you plate? 100ul? 250ul? The entire transformation volume?

Tom Masi

So I have previously transformed using 2 ul of ligation mix and still never got colonies. That is why I increased it to 5 ul.

I ligate 50 ng of vector and a 1:3 ratio has 37.2 ng of insert.

I plate 200 ul (x2) from a 500 ul SOC mix after the 1 hr incubation in 2 plates each. I have even tried to spin down the cells and resuspend and plate.

Thanks for the information. Sounds like you have a problem with the ligation reaction itself.

Here are a few things you might want to try different (and what I have always done with success):

1. Perform the ligation at RT O/N.

2. Cut the amounts of vector/insert way back - use 1ng of vector and then adjust your vector ratios based on that.

3. Increase you ratios to 1:40, 1:50, 1:60 - never found the 1:1, 1:3, 1:5 ratios to work consistently.

4. Use 1ul for transformation.

5. Resuspend transformed cells in 250ul SOC and after incubation plate the entire amount.