pDR244 Cre Ts枯草芽孢桿菌無痕敲除（cre-lox敲除）載體質粒

cre/lox-Mediated Loop-outProtocol for pDR244

(1)Transform pDR224 into one of the Bacillus subtilis BKE collection or any otherstrain harboring a

loxP--ankedantibiotic resistance cassette with selection for spectinomycin resistance at30°C

(2)Transfer several transformant colonies to plain LB plates and incubateovernight at 42°C

(3)Screen resulting colonies for plasmid curing (spectinomycin sensitivity) andthe antibiotic

resitancecassette (erythromycin sensitivity).

(4)Streak for fresh single colonies and then confirm loss of the cassette by PCR

Transformation of plasmid DNAto competent E. Coli cells

1. Thaw competent cells on ice. 20–200μL per tube

2. Add max. 1-3μL of plasmid

3. Mix very gently!

4. Incubate the tubes on ice for 30 min

5. Heat shock the cells for 45 sec to 2 min at 42°C

6. Place the tubes immediately on ice for at least 2      min

7. Add 800μL of SOC medium to each tube

8. Incubate for 1 hour at 37°C and shake vigorously

9. Spin down briefly and remove most supernatants

10. Resuspend cell pellet with the rest SOC medium in      the tube by pipetting

11. Plate out the suspension on a LB agar plate      containing the appropriate antibiotic. Incubate the plates overnight at      37°C