Why are there different sizes of inserts being obtained after cloning despite the same enzymes being used for restriction digestion?

05 May 2014 1 3K Report

Different colonies resulting from the same innoculant were used for plasmid isolation and the sequence cloned is from a plant virus vector used is pTZ57.

Muhammad Faraz Bhatti

First of all, If it is only one PCR product that you have ligated and is product is gel purified then there are slim chances of getting multiple inserts, however, if the ligated product is a smear then it is quite possible there are multiple products in it.

Secondly It is quite possible that the ligated product have got the same restriction site within the clone that may result in multiple products.

Thirdly the plasmid DNA might be sheared

I hope this might help

Cheers

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