Hi Tasnim, how did you delete the undesired DNA? Did you use restriction enzyme(s)? if yes. It would be easy to make your plasmid circle again especially if one restriction enzyme was used.

Hi,

If you are building your plasmid by PCR, remember that most oligonucleotides produced for use as PCR primers do not have a 5-prime phosphate unless you specifically order modified PCR primers. Most companies offer this option. Also Taq polymerases tend to add an additonal adenine on the end of PCR products, which allows for TA cloning:

https://www.thermofisher.com/us/en/home/life-science/cloning/ta-cloning-kits.html

Terry

Dear Tasmin

you can use PIPE cloning which is very similar to infusion but do not require any specific kit, just 2 primers for plasmid pcr inducing n terminal sequence that overlap, an high fidelity polimerase and mach1 e.coli cells.

you can find more information about PIPE on my blog: PROTEOCOOL at page 1 : https://proteocool.blogspot.com/p/cloning.html?m=1

ProteoCool n°1 (Cloning methods overwiev )

and

ProteoCool n°3 (Primer Design: PIPE Cloning )

good luck

Manuele