DNA Ligation Not Working Troubleshooting

[gandalfthegrey]

Hi all,
My ligation is not working.

I am trying to subclone an insert from one construct to another plasmid.

Using T4 NEB and used different ratios and ligation incubation times. I have also tried highly competent bacteria to no avail.

please help me!

[dnamolecule]

Hello there,
I tend to do my ligations with less DNA as it may be toxic to bacteria:
Try using 50ng vector and ~3-fold molar excess of insert.

I am almost always doing 3-way ligations which is why I use so much DNA. I think you should be able to get away with less but these amounts work reliably for me.

Try this formula with 50ng vector:



Cheers