Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

self ligation - (Jun/06/2013 )

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.