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Engineering Cys2His2 Zinc Finger Domains 3277. Put tubes on the wheel, 37°C for 90 min.8. Plate 300 µL of the actual mating from step 6a above (CSH100 transformants+ KJ1C) on a LB/TK plate and on a LB/TKS plate. Spot 5 µL of each of thenegative controls on a LB/TK plate and on a LB/TKS plate. Incubate all platesovernight at 37°C.As shown in Fig. 5, only KJ1C cells harboring the desired double recombinant F′should grow on LB/TKS plates. All the matings are plated on LB/TK plates as wellto check that the counter-selectable marker (sacB) is working to eliminate KJ1C cellsharboring single recombinant and nonrecombinant F′s (see Fig. 5). (Note thatexpression of the sacB gene is lethal in E. coli cells when they are plated on mediumcontaining sucrose.) Typically, at least a 10-fold reduction in the number of coloniesis observed when comparing the number of colonies on LB/TK with the number ofcolonies on LB/TKS plates.9. After KJ1C cells harboring the desired double recombinant F′ have been identifiedon the basis of growth on LB/TKS plates (see Note 8), clonal isolates have beenpurified by restreaking candidates two times to LB/TKS plates. (Note: KJ1C cellsgrow quickly, so if they are streaked early in the morning, new pickable colonieswill typically be ready by evening to streak again.). Typically, two candidates arechosen (which are designated “A” and “B”) for each desired clone because a smallpercentage of cells that survive on LB/TKS plates will fail additional subsequentverification tests (as described next).3.1.3. Selection Strain Verification3.1.3.1. GENETIC CONFIRMATION OF F′ TRANSFER1. Resuspend each colony (A and B candidates) in 100 µL of 1X M9 Salts (this istypically done in wells of a sterile 96-well plate). Spot 5 µL of each cell suspensionon an M9 minimal medium plate (see Note 9).2. After overnight growth at 37°C, verify growth of cells within the spots. Candidatesthat fail to grow should be discarded.3.1.3.2. SEQUENCING OF THE RECOMBINANT F′ REPORTERFor candidates that successfully demonstrate growth on M9 minimal mediumplates, the portion of the F′ harboring the target DNA site is amplified andsequenced to verify the reporter.1. Use 20 µL of the cell suspension from step 1, Subheading 3.1.3.1., to inoculate a4 mL LB/kanamycin (30 µg/mL) overnight culture.2. Transfer approx 100 µL of saturated overnight culture to a 1.5-mL microcentrifugetube. Spin at maximum speed for 1 min in a microfuge. Remove media using apipet tip and then resuspend cells in 100 µL PCR-grade ddH 2O. Heat the cell resuspensionat 95°C for 10 min and then spin at maximum speed for 1 min in amicrofuge. Remove 50 µL of the supernatant to a fresh tube and use this crudepreparation of genomic DNA as template for a PCR reaction as follows: