Hi.

Is your ligase enzyme and buffer functional?

What molar ratio and mass are you using for ligation?

Yes, My ligase enzyme and buffer and working very well as I have checked the liagtion mixture of gel and proper ligation DNA band has appreared. The molar ratio of (1:10) for double digested vector(59.2 microgram per microlitter, A260/280 = 1.90) and double digested insert of 62.9 microgram per microlitter, A260/280 = 1.85) was added.

Also, what is the amount of vector that you are using for the ligation, and the size of vector and insert?

Additionally, are you adding the correct concentration of antibiotics to the agar plate after transformation?

Many times, we miss out on the minute mistakes that we could be doing.

The size of my vector in 5.3kb and insert is 800bp, I am adding vector and insert in 1:10 ration, respectively. The kanamycin we're using it in 25 microlitter for 25ml of LA plate. I am also using recently opened kit for buffers and enzymes.

What is the amount of plasmid in ng you are taking for ligation reaction?

And, what is the volume of your ligation mixture?

I recently used 120 ng of vector for 20 microliters of reaction in 1:8 ration of vector and insert respectively.

I think you should go for a 10ul reaction instead of 20.

You can also try increasing the vector mass till 300 ng, and see if it works out.

Sure. I'll check and let you know