self ligation - (Jun/05/2013 )
i Have a vector(pF12a RM flexi vector) and i want to use this vector as a negative control for my cloning. i mean without an insert.
this vector has a barnase gene at the MCS site which need to be removed by Sgf1 and Pme1 restriction enzymes.
so now if i do restriction digestion, i will end up with flanking cuts in my vector and for the clonging i need to ligate these ends, but as these ends being non compaitable or being different restriction site ends, how can i ligate the vector and use as a negative control for my cloning.
to summorize, i want to know how i can make a vector to self ligate that has different restriction sites on either ends of it.
Later i want to use these in my transient trasfection.
Please anybody help me .
Thanx in advance.
Just to make sure that I have this correct - you are looking for a positive control for ligase activity?
You currently have a plasmid that you have double cut with SgfI and PmeI which produce non-compatible ends that you will use in the cloning of a particular insert.
If this is the case - and you just want a control for the ligase, you can just single cut the plasmid with one of the restriction enzymes and use that as the control.