Any standard plasmid DNA purification can be used as long as a phenol/chloroform extraction and subsequent ethanol precipitation step are employed. We have found that Qiagen DNA column purifications may not be completely digested unless the DNA is phenol/chloroform extracted and ethanol precipitated. This protocol is for 10 µg DNA but can, of course, be scaled up. All parts of the protocol should be followed exactly.
- Digest plasmid with Xba I restriction endonuclease (NEB #R0145):
Plasmid DNA (at least 640µg/ml) 10 µg
NEBuffer 4 10 µl
BSA (10 mg/ml) 1 µl
XbaI(40 units) 2 µl
H2O to 100 µl
Incubate overnight at 37°C.
- Nick the XbaI-linearized vector with Nt.BbvCI (NEB #R0632) by adding 2 µl (20 units) of Nt.BbvCI (10,000 units/ml) to the above reaction and incubating for 1 hour at 37°C.
- Purify linearized, nicked vector by phenol-chloroform extraction. Resuspend in 100 µl of TE buffer.
- Determine vector concentration. Dilute to 20 µg/ml final concentration as an alternative to the pNEB206A cut vector in the USER Friendly Cloning Kit Protocol.