Hi Arihant,

maybe you can give some more inforation about your cloning strategy and the enzymes you are using?

One obvious reason why you don't get you band (if you have cloned sucessfully) is that one of your digestion enzymes is not working (even if you think that the vectorband has the same size as before cloning it is - depending on the size of the vector and the insert - sometimes difficult to distinguish between empty vector and empty vector + insert) - try new restriction enzymes and/or restriction sites which are flanking the restriction sites you used for cloning (if possible).

Another reason might be that sometimes one restriction site is destroyed during cloning or not working anymore due to methylation (depending on the restriction site and the flanking sequences) - try restriction sites which are flanking the restriction sites you have used for cloning (if possible).

Another possibility is that you didn't clone successfully your insert but that you have a insert contamination in one of your reaction components (if you are unlucky even you pipette can be a source of contamination).

Did you include a negative control/no template in your PCR?

If you haven't done this already I would recommend to repeat your PCR with a negative control to rule out contamination.

And I think it would be a good idea to sent your plasmid for sequencing.

Good luck

What's the size of your insert and your vector? If it's a small insert, you might not be able to see it on an agarose gel. Band brightness is based on amount of DNA & it's harder to see 10,000 copies of 100bp than it is to see 10,000 copies of 10,000bp.

Just sequence your plasmid - you'll want to check for any changes anyway. No one uses restriction digests to verify inserts anymore. Sanger is faster, cheaper, and more accurate

It is possible that your plasmid prep is a mixture of empty plasmids and plasmid with your insert. Even if only a tiny fraction carried the insert (less than 1%) you would still get a strong signal after PCR but you would probably not see the insert on a gel after restriction digest.