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Restriction enzyme - (Dec/20/2017 )

Hi everyone

I've just wondered: May I use only one restriction enzyme (such as BglII), to introduce a gene into a plasmid? How can I know if the gene direction is converted when in ligated into the plasmid? (please see the picture attached below)

Thank you for answer my question


Yes you can use just one RE. The difficulty, as you say, is that then you can have problems with the direction of the insert. To overcome this you basically just need to screen for direction of your insert - you can do this in a number of ways, but the most common are sequencing and using a primer that is anchored in the plasmid (forward primer somewhere 5' of the insert) and one in the insert (reverse). This should only give you a PCR product when the insert is in the correct orientation.