Have you looked to see that you recovered plenty of DNA after purification? Especially of the insert?

Also have you done control ligation with only the single fragments from each to see which one is contributing most of the background colonies? Then maybe you can figure out some strategy to reduce that background like digesting with an enzyme that only cuts in the stuffer fragment after ligation.

Hi Emilian

in addition to what Michael J. Benedik suggested I would recommend to do also a transformation of digested and purified vector and fragment without ligation to see if you get also colonies there.

If you have colonies in your no-ligation control (esecially if it is similar to the amount of colonies you get with ligase present) you clearly have a problem with uncut vector.

In this case I would recommend to increase your digestion time and to consider to run your gel longer before cutting out your bands and maybe also use a lower percentage gel (especially your 7,2 kb Car-T vector before digestion and your 5,7 kb Car-T fragment after digestion might not seperate very well and as you usually will have only tiny amounts of uncut vector you will not see this on the gel).

You might also consider to make a transformation with the water and the bufffer you use to exclude that you reagents and your pipette are contaminated (not very likely but it can happen).

If you have excluded/solved the problem with uncut vector I would advice in addion:

Be fast when your cutting out your bands if you use UV radiation to make your DNA visible. UV radiation will damage your DNA and can greatly interfere with your cloning efficiency.

Try ligating at lower temperatures for longer time (e.g. 48 h at 4°C)

Try different ratios of vector to insert

Do not use to much DNA for your ligation reaction (around 25 ng should be enough)

Consider using a new batch of ligase

Good luck

My suggestion will be:

1. Design primer pair with restriction sites to PCR amplify the smaller fragment from shx_b plasmid, then digest with NheI-HF, SphI-HF, and Dpn1 (to digest the plasmid template; provided the plasmid was amplified in E. coli before, and so methylated).

2. Digest CAR_T plasmid with NheI-HF, SphI-HF, and CIP (to dephosphorylate the plasmid template so that it would self-ligate back), then gel extract the bigger fragment

3. Then ligate the smaller insert and bigger plasmid fragment together.

Second option:

1. Find additional restriction sites in the unwanted fragments of both plasmids and digest, to reduce the chance of re-ligation.

All the best!