Digestion of Plasmid
Posted 09 January 2002 - 07:35 PM
I recently subcloned CaM Kinase Iv in to a plasmid.
I did the mini prep using the standard "molecular cloning-protocol".
However it seems that the digestion is not proper.
I mean I find it difficult to distinguish the circular plasmid & supercoiled plasmid Vs vector & insert.
Moreover, while pipeting 1.5micro litre of Xho-I enzyme, I find it very clumsy.
How can I circumvent this situation?
Looking forward to your advise!
Have a nice day.
Posted 14 February 2002 - 07:51 AM
1) are you SURE that your subclone plasmid contains TWO XhoI-sites?
2) make a dilution of XhoI so that you can comfortable pipette the enzyme (e.g. 0.5 U/microliter)
3) digest 1 microgram of your plasmid with 3 U XhoI, do not forget BSA!; in 30 microliter end-volume, 37C, 2-4 hours
4) load as control in an agarose gel: in one slot the linearized vector (without insert), in the next slot your insert (without vector), in the next slot your digested plasmid DNA (kinase subclone), in the next slot your circular (not digested) plasmid DNA (kinase subclone) and finally, in the next slot DNA-marker (e.g. 1-kb-ladder)
5) make two photos (one after halftime of the electrophoresis, and one at the end) of the gel to get detailed infos about the fragments
6) if the digestion is not pretty, purify the plasmid DNA!
7) vary the agarose concentration to see notedly the bands if the insert has similarly the same size like the vector; eventually use another enzyme that cut in a different pattern