Problems with pX459

[Bin Gu]

Hi 

    I've just got the pX459 plasmid from Addgene. But got into some problem when digesting with BbsI, it always became smear after digestion. I cut pX330 simultaneously and run on the same gel and that's fine( as the photo I posted. Did anybody run into the same problem and have any suggestion?

Bin

[Bin Gu]

Sorry, it's pX459 BbsI on the photo.

[Cor]

Hi Bin,

I ran in the same problem with pX330, I isolated 2 fresh mini's (spin column) and I pooled both mini's and gave them an extra phenol and phenol chloroform extraction plus an ethanol precipitation, this seemed have solved my problem.

Good luck,

Cor

[Joey Riepsaame]

Hi Bin,

I have experienced the exact same thing. Apparently, FD-BbsI (Fermentas) completely cuts up relatively "unpure" DNA, for example DNA obtained from mini-preps. I never have this when I use DNA obtained from MIDI preps. Like Cor said, try an additional phenol/chloroform extraction.

BW,

Joey