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Extra resources for Applications of Chimeric Genes and Hybrid Proteins Part A: Gene Expression and Protein Purification
8. As lysis occurs, add 100/xl of chloroform to each tube. Note: After 6 hr, add chloroform, irrespective of whether there is obvious lysis (occasionally, high-titer lysates can be obtained without apparent lysis). 9. Vortex the tubes briefly and then centrifuge at 4500g for 10 min. 10. Transfer the supernatant to sterile screw-cap tubes (taking care to avoid transferring any of the chloroform) and store in the dark at 4 °. , they have picked up the plasmid encoded lac fusion) and some of which are nonrecombinant Lac- parental phage.
Incubate the tube at room temperature for 5 min. 5. A d d 1 ml of LB broth containing 10 mM MgSO4. 6. Incubate the tubes at 37 ° for 1 hr. 7. Pellet the cells at 4500g and decant the supernatant. 8. Wash the pellet with 1 ml of 10 mM MgSO4, vortex briefly, and pellet the cells again. 9. Decant the supernatant, vortex the tubes to resuspend the cells in the residual liquid, and plate the entire contents of each tube on a minimal lactose plate [in the case of A(gal att bio) strains, the plates must be supplemented with biotin to a final concentration of 1/xg ml-1].
Potential lysogens should be chosen from the plate in which the fewest number of phage was used in the infection. Further steps to avoid multiple lysogens are described. Targeting Lac Fusions to the Chromosome 1. Prepare 10 2, 10 4, 10 6, and 10 8 dilutions in T M G buffer of the lysates described earlier. 2. 5 ml of 10 mM MgSO4. 3. In a small culture tube, mix 50/xl of cell suspension with 10 t~l of each dilution of the A lysates. 4. Incubate the tube at room temperature for 5 min. 5. A d d 1 ml of LB broth containing 10 mM MgSO4.