As my desirable insert size is 490 bp but using M13 primer for plasmid amplification I got different band sizes. why there are different types of bands for the same?
Are you certain that your insert has been cloned into the plasmid? You have loaded so many samples, are these amplified using same plasmid or different plasmids? Different sizes are usually observed when there is unsuccessful ligation.
i am sure that the insert has been ligated. These are ligated with same plasmid Sir.
What do your controls look like? It's entirely possible for plasmids to self-ligate with no insert. What size band would you expect to see for just M13 primers & no insert present?
I'm also not convinced that you are getting amplified products in most of those lanes.
First of all Did you confirmed the clone by restriction digestion?
Second thing if you perform colony PCR there will be some false positive results.
Check your primer pair and dilute it again from your stock before performing PCR.
Conventional PCR is not the standard method to check successful cloning you should go for the restriction digestion and Sanger sequencing.
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