Conjugation using ET12567/pUZ8002

1. Transform ET12467/pUZ8002 with oriT-containing vector, and select for the incoming plasmid only
2. Inoculate a colony in to 10 ml LB containing Cm, Km and antibiotic used to select for the oriT containing plasmid and grow overnight. Chloramphenicol and Kanamycin sensitive segregants arise frequently among transformants, and therefore it is recommended that more than one culture is set up.
3. Dilute the overnight culture 1:100 in fresh LB + antibiotic selection and grow to an OD600 of 0.4-0.6.
4. Wash the cells twice with an equal volume of LB (to remove antibiotics) and resuspend in 0.1 volume of LB
5. While washing the E. coli cells, for each conjugation add approximately 108 Streptomyces spires to 500 µl 2 x YT broth, heat shock at 50 oC for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.
6. Add 500 µl of E. coli cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and thn resuspend the pellet in the residual liquid.
7. Plate out on MS agar + 10 mM MgCl2 and incubate at 30 oC for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9.
8. Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30 oC
9. Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.