you could check the competency of the cell, and type of the cell because your amplicon size is 9kb , if possible use another strain for transformation.

Please provide a better explanation of how the DNA is generated? If you are merely amplifying a linear molecule by PCR without any digestion or treatment of the termini, then your 5' ends are not phosphorylated and will not ligate.

Thank you for your answer. I simply amplify the DNA fragment using PCR and Gel purify it using the final product as the template for ligation. What type of treatment do you suggest for efficient self circular ligation?

Thank you

Tasnim Reza, the problem is that PCR product is not a substrate for ligation. You need to have a terminal phosphate on the 5' end of DNA for ligation to occur. Since the 5' ends are the DNA primers, they are not phosphorylated and hence not substrates for ligation. When you ligate to a digested plasmid, you have one 5' end from the plasmid which carries a terminal phosphate, so that can ligate (although not efficiently). You have a few options, one would be to redesign your PCR primers to have a restriction site at the ends and then digest the PCR product and ligate. The digestion will reveal a 5' phosphate residue. This is probably the easiest to do if your construct can tolerate the addition of the 6 bases to create a restriction site.

The second option would be to take your PCR product and perform a phosphorylation reaction using ATP and polynucleotide kinase. This will then be a suitable substrate for ligation.

A third option would be to also design new PCR primers that overlap so that the linear PCR product have overlapping ends. If the ends are suitably long (20-30nts) you can just ligate directly and the cells will create circles without ligation.

Thank you so much for the information. I have a question.

I successfully cloned my insert using the same PCR product. Then why is it not closing on itself? Is the procedure different?

Because the vector is suppling terminal phosphates for ligation which are not present on only the PCR product.

Thank you for your answer.

The vector I used was the same PCR product I am talking about. The vector is the one I am trying to close to make a control plasmid without the insert. So for my cloning experiment may be my insert (which was also a pcr product) might have had 5' ends.

So If I treat the vector with polynucleotide kinase it should be able to close on itself?

So it depends upon the vector and how it was linearized and treated. Please provide more information if you want some advice.

Thank you for your help. Below are the steps I followed for previous cloning which worked.

1. The Backbone vector was amplified using PCR reaction.

2. The reaction was run on Agarose gel and the band was excised to get the vector.

3. The insert was in a PUC plasmid. The insert was isolated the same way as the vector (PCR then gel)

4. After purification of the bands Infusion HD cloning kit by clontech was used to put the insert in the vector.

5. The reaction was then transformed in stellar competent cells.

The cloning worked and confirmed by sequencing.

So that time I have not treated the vector with PNK yet it worked. But when I am trying to close the vector without any insert I am not getting any colonies/ empty colonies.

I tried another control experiment where I cut my vector plasmid with SMAI and ECORI went through the same process and SMAI did not have any colony but ECORI did.

So I think it is something to do with the blunt end ligation.

I would agree that you are having troubles with blunt end ligation if you can not ligate a SmaI digested plasmid. However if as you write you are making your vector DNA by PCR amplification then it will not ligate without further treatment. Infusion Cloning uses an entirely different method, similar to Gibson assembly, to put your DNA molecules together.