I am cloning my guide RNA in pLenticrisprv2 plasmid. I picked colonies and did primary culture to miniprep. after miniprep i did a pcr using vector specific (hU6 promoter) forward primer and my bottom oligo as reverse primer. I got two different type of bands from different colonies. while the specific band should be around 275bp which i get in one colonies, other two colonies gave band of around 175bp. moreover when i sequenced the two colonies that gave 175bp there was no guide RNA insert and a part of plasmid was missing from in between suggesting size of eluted plasmid to be of 8Kb while it should be around 12kb.

Kindly suggest if there is no insert then why did I get band?

(in the picture attached colony 1 and 3 gave non specific band while colony 2 gave specific band. Colony 2 is yet to be sequenced.

Thankyou.