REFERENCES: Faelen et.al. 1978. CHS Symp. Quant. Biol. 43: 1169-1177; Van Gijesegem and Toussaint. 1982. Plasmid. 7: 30-44; Seeberg and Wiedemann. 1984. J. Bacteriol. 157: 89-94; Kado and Liu. 1981. J. Bacteriol. 145: 1365-1373.
 

MATERIALS: (for parts A and B)

1. 5 ml broths (4)
2. Nitrocellulose discs (47 mm), sterile (2)
3. Broth plates (at ambient temp.) (2)
4. Minimal salts plates (3 for part A, 6 for part B)
5. Minimal salts solution (50 ml plus 6 x 9 ml in McCartneys)
6. Side-arm flasks containing 15 ml of broth (4 x 100 or 125 ml)
7. Scalpel, forceps, ethanol
8. Spectrophotometer (A 546), shaking incubators (32/42o, 370C)
9. Eppendorf centrifuge

METHODS: (based on ref. 3)

A. PREPARATION OF R' PLASMIDS

1. Grow wild type gene donor strain (autotrophic for other markers) (e.g. S. typhimurium P9003 for his+ DNA) and RP4::mini-Mu (pULB113) donor strain (P3140 is MXR/pULB113 of ref. 2) O/N in broth at 37 C with selection (P9003 = Smr3140 = Ap, Km, Tcr.
2. Regrow both cultures to A546 = 0.8 in side-arm flasks at 37ºC. Mix 5 ml of each culture and pellet cells in a McCartney (or Lennon/Star/Harrison) bottle. Resuspend cells in 50 µl of broth by thorough vortexing.
3. Place a sterile nitrocellulose disc (47 mm diameter) on a broth plate (dried and prewarmed to ambient temp.). Pipette the entire cell suspension onto the disc. Spread cells over the disc with the pipette tip or a bent, flamed pasteur pipette to hasten drying. Incubate at 37ºC for 3-5 hours.
4. Use a flamed scalpel to slice disc into strips about 1 x 1.5 cm (e.g. 8 pieces) and flamed forceps to transfer strips into 10 ml of minimal salts or 10 mM MgSO solution in a MCCartney bottle. Vortex bottle 4-5 times over a 15 - 20 min. period, tip S/N into a second bottle, rinse strips with 5 ml of min. salts and combine rinse with first suspension. Resuspend cells in 10 ml min. salts, dilute by 10304d 105 min. salts and store remainder at 4ºC. Plate 0.1 ml of each dilution on minimal agar plates containing Sm (for P9003), Ap, Km and Tc, and supplemented with the growth requirements of the gene donor strain (e.g. P9003) but not those for P3140 growth (Sm selection alone is usually not satisfactory). Expect about 10 - 100 colonies on the 10 dilution plate - i.e. about 10-10 exconjugants per recipient. Colonies take at least 24 hours at 37ºC to come up. These are the R' donors.

B. SELECTION OF SPECIFIC R' PLASMIDS

1. Grow a checked exconjugant (R' donor) from the mating in part A O/N at 32ºC in broth containing all appropriate antibiotics (i.e. Ap, Km Tc and Sm for P9163 from a "P3140 to P9003" mating). Grow the tester strain (which has a mutant allele of the gene to be cloned, growth requirements other than those of the R' donor and ideally the recA mutation) O/N in broth at 37ºC.
2. Regrow the R' donor to A546 = 0.5 at 32ºC then incutate for 2 hours at 42ºC with good aeration to induce mini-Mu transposition. Regrow the tester strain to A546 = 0.8 at 37ºC.
3. Conduct the mating as in part A above using 5 ml of each regrown culture. Plate 0.1 ml of the washed mating mixture undiluted (10) and after 10-fold dilution (10) in min. salts. Also, plate 1 ml of the washed mixture by resuspending it in 0.1 ml of min. salts. Use minimal plates which counterselect both parental strains but not tester cells which have received an R' carrying the desired gene. Dilute the mixture by 10, 10 and 10 and plate 0.1 ml on the same plates supplemented with the product of the desired gene to estimate the frequency of R' transfer to tester cells (should be about the same as for pULB113 transfer). Expect a given gene to be transferred at about 10; to 10; per tester cell (i.e. about 1-100 colonies on the 10 plate.
4. Incubate plates for 36-48 hr. at 32ºC.

RecA strains grow better in 1 - 2% sucrose.

It may be useful to monitor the plasmid profile of starting and derived strains during these procedures by running plasmid mini-preps (from 1 ml of O/N cultures using the method in ref. 4) in 0.8% agarose gels. The R' plasmids will be 100-150 kb in size.