ensure that your construct is in frame with your vector. Also that you are using the appropriate antibiotic. Sometimes we can oversee minor details like this.

Did you sequence to verify your vector sequence is what you want? Or at least run out an aliquot on a gel to check the size & concentration?

Every time you do a digest, cleanup, etc. you'll lose some of the starting product. It's possible you might not have enough plasmid left for the transformation to work.

Thankyou Amina,

I did ensure that my construct is within the vector after ligation by doing AGE, and antibiotic is kanamycin I am using it in proper proportion only

Thankyou katie,

I did verify my vector after digestion by running both uncut and the cut vector on AGE concentration was good I did check the concentration in uv spectrophotometer too and used neb website for ligation reaction based on the concentration I got in uv spectrophotometer

Try a fresh tube of the DNA ligase and set up an overnight ligation. I know, it's possible to do a "5 minute ligation", but you'll get better results with a longer incubation step.

Did you remember to phosphatase the vector before the ligation? Otherwise, the most favorable reaction is for the vector to just re-ligate back together without your insert.

Have you done any insilico cloning just to confirm your gene of interest is within the frame? Looks like the problem is with your ligation step. Increase the ligatiion time and use a fresh Ligase.

Thankyou katie

I did phosphotase my vector I too have a feeling like problem is with ligation so I'll try overnight ligation once at 16C

Hello Abir thankyou for your response

I did insilico cloning have a feeling might be a problem with ligation so I am changing the enzyme and gonna try again with 16C overnight incubation and will come back to you