To successfully use the CloneJET PCR Cloning Kit from Thermo Fisher for ligation and transformation in E. coli cells, follow these general steps:

1. PCR Amplification: Amplify your DNA fragment of interest using PCR. Be sure to incorporate the appropriate restriction enzyme sites in your primers for subsequent cloning.

2. Purification: Purify the PCR product to remove primers, nucleotides, and enzymes. This can be done using a DNA purification kit or method of your choice.

3. Digestion: Digest the linearized vector (provided in the CloneJET kit) and the PCR product with the appropriate restriction enzymes. This will generate compatible sticky ends for ligation.

4. Gel Electrophoresis: Run a gel to confirm the presence and size of your digested PCR product and vector.

5. Ligation: Mix the digested vector and PCR product at a suitable molar ratio (typically 1:3 vector to insert) in the presence of ligase and reaction buffer. Incubate the ligation reaction at the recommended temperature and duration specified in the kit's instructions.

6. Transformation: Transform the ligation mix into competent E. coli cells according to standard transformation protocols. The kit may include competent cells, or you can use your own.

7. Plate and Incubate: Plate the transformed cells on selective agar plates containing the appropriate antibiotic. Incubate the plates overnight at the recommended temperature for E. coli growth.

8. Colony Screening: The next day, examine the plates for bacterial colonies. Pick colonies and perform colony PCR or minipreps to verify the presence and correct size of the inserted DNA.

9. Sequence Verification: If necessary, verify the correctness of the cloned insert by sequencing.

It's important to follow the manufacturer's protocol and guidelines provided with the CloneJET PCR Cloning Kit for the specific details of each step and any additional recommendations. Always perform controls and replicate the experiment to increase the chances of success.

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