Cloning a gene with XhoI & EcoRI gives bands 700, 500, and 2800. Insert size?
When cloning into vector flanked by XhoI and EcoRI, complete digestion releases backbone and insert fragments separated by electrophoresis. Backbone appears as largest band, here 2800 bp, while insert may be cut internally into smaller pieces if it contains internal recognition sites. Observing 700 bp and 500 bp bands plus 2800 bp backbone indicates insert of 1200 bp split into two subfragments by internal site. Summing subfragments gives insert total length. This approach confirms successful ligation, estimates insert size, alerts to unexpected internal sites affecting directional cloning, an
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.
