Digestion dilemma - (Oct/12/2007 )
I have one pDNA to cut with two restriction enzymes,KpnI and XhoI,both are from the Fermentas.The problem is that the KpnI is the conventional enzyme with its unique buffer and Xho I is a FastDigest with its own FastDigest buffer.How can I cut the pDNA with the both enzymes?Is it possible to cut using one buffer in one reaction mix
or Do I need to cut separately?Is anybody there to help me???
I would not do a double digest. I would first digest with XhoI then celan up with an agarose gel clean up kit (I use the Wizard® SV Gel and PCR Clean-Up System) but without an agarose gel (after the first cut you should only have linearized product, right?). Then after the second digest I sould clean up again with this system but this time with an agarose gel.
I would do a double digest in NEB buffer1. Which buffer system do you use? Fermentas? Is it possible to get yourself some NEB buffer1 .
yup,I have NEB buffer 1 but the main thing is that these both enzymes have their different buffers like I mentioned in my original post.You mean,instead of that I can use the NEB buffer 1 only for the reaction.How???
Check the components from all three buffers.
Check the activity of you enzymes. If one has 100 % activity and other enzyme has 50-75% activity in a particular buffer, NEB recommends that you can double the amount of the lesser active enzyme for maximum digestion.
The other way is to check the composition of your buffers and do sequential digestion starting with the buffer with lower salt concentration for first digestion and then increasing the concentration for second digestion.
Hope this helps.