Need help troubleshooting digest. - (Apr/05/2012 )

I'm trying to replicate a method listed here <http://www.jbc.org/c.../15607.full.pdf>. I want to create a deletion mutant, and I've modified the original technique a bit.

I obtained pBluescript-cnf1 (pB-cnf1) vector via miniprep of old freezer inventory(JM101-pB-cnf1). Instead of km (pUC4 has been commercially discontinued), I've decided to utilize chloramphenicol resistance, so I've digested pKD3 (GenBankAY048742.1) and pB-cnf1 both with XbaI. Note: I used NEBCutter to identify XbaI cut sites. According to NEBCutter there are only 2 XbaI-recognition sites in pKD3.

The gel for digested pB-cnf1 shows a single band indicative of a single cut, but the gel for digested pKD3 showed three instead of two: one is indicative of cm plus flanking regions (~900bp), second is indicative of the remaining pKD3 (~1850), and the third is indicative of undigested pKD3 (2804). I'm using FastDigest XbaI/buffer (Fermentas) which calls for a 5 minute incubation for digest. I've run 3 more digests, with increased incubation times (10, 15, 20) to still see three bands.

I went ahead and ran a ligation reaction (pKD3, pB-cnf1) and transformed it into DH5alpha with no luck. I'm assuming the issues I'm observing in the digest need to be addressed in order to have a successful ligation. Any suggestions or advice?

Try transforming the plasmid into a dam- strain, the incomplete cut may be due to methylation.