Dear Trupti Tayalkar

i never used the pDirect23A and the AarI enzyme and maybe i loss some point, but i do not understand why to insert a so short region (23bp) you are perfromin restrction/digestion and ligase approach which is complex and not so simple to do with a so small fragment.

I suggest to you to perform a vector PCR adding in the primers regions overhanging that contain the sequence of the region to be inserted.

you can see an example of something similar on the following link:

https://proteocool.blogspot.com/2021/09/tips-of-week12-rapid.html

regarding your specifci problem, i have some doubt:

1) why are you using a single digestion site?

2) did you perfrom plasmid defosforilation after digestion?

3) did you produced also a 23bp rna sequence flanked by AarI digestion sites? or did you ligated the 23bp itsefl?

Since with a single digestion site, you can have:

- Plasmid auto re-ligation:

- ligation of the insert in the opposite direction:

best regards

Manuele

I have selected unique restriction site in the plasmid i. e. AarI

I have used fast AP alkaline phosphatase after digestion

I have synthesized a 23bp sequence which contain 20 bp gRNA and the flanking region (3bp) by@@ AarI digestion site