Gene Disruption using pMAD plasmid
Construction of plasmid.
Transform RN4220 using the plasmid. Select on erm 2.5ug/ml+X-gal (50ul of 40mg/ml).
Incubation at 30oC. Pick blue colonies to check for plasmid.
Isolate the plasmid and use it to transform RN6390.
Select on erm 2.5ug/ml+X-gal (50ul of 40mg/ml).
Incubation at 30oC. Pick blue colonies to check for plasmid.
Pick a blue colony, grow overnight at 30oC in TSB with erm (2.5ug/ml).
Plate the dilutions on TSA+erm 2.5ug/ml+X-gal (50ul of 40mg/ml).
Incubate at 44oC.
Pick light blue colony, grow overnight at 30oC in TSB (no antibiotic added).
Plate the dilutions on TSA+X-gal (50ul of 40mg/ml).
Incubate at 44oC.
Pick white colonies. Patch on TSA+erm 2.5ug/ml and TSA+X-gal (50ul of 40mg/ml)
plates. Select white clones from X-gal plate which are not growing on erm plate.
Check with PCR for right integration.
PCR Method
Take 500ul O/N culture. Spin down cells at 5k/5min. Discard the supernatant.
Suspend the cells in 200ul EB or TE.
Add equal volume of beads. Bead beating at max speed for 40-60 sec., twice.
Spin at 12K for 10 min.
Use 2.5ul of supernatant as template for PCR. (use it within an hour to avoid degradation
of DNA).