Transformation of BAC into Recombinogenic Strain SW102
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Make a 5 ml o/n culture either from the SW102 freeze stock or from a single colony.
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Dilute the o/n culture 1:50. Incubate with appropriate antibiotic selection in at 32°C shaking water bath until the density reaches an OD600 of 0.6.
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Cooled down the SW102 culture in the ice/water bath slurry and subsequently transferred into pre-cooled 14 ml Falcon Round-Bottom tubes.
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Spin down the bacteria at 0°C, 4000 RPM for 5 min.
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Pour off the supernatant and briefly invert tube on a paper towel, and add 1 ml ice-cold ddH2O. Leave the tube on ice for 3 min and resuspend the pellet in the ddH2O by gently swirling the tube in the ice/water bath. Add another 9 ml ice-cold ddH2O and pellet the samples again.
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Pour off the supernatant, resuspend as in step 5 and spin twice more.
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Remove all supernatant by inverting the tube on a paper towel. Gently resuspend the pellet as before to a final volume of 50 μl.
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Mix 25 μl of the freshly made electrocompetent cells with 100ng BAC DNA. Transfer the cocktail to a pre-cooled 0.1 cm cuvette.
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Transform by electroporation at 25 F, 1.75 kV, and 200 ohms, twice.
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Transfer the bacteria to a tube with 1 ml LB medium. Incubate at 32°C for 1 h.
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Plate serial dilutions (100 μl, 100 μl of a 1:10 dilution, 100 μl 1:100, and 100 μl 1:1000) the transformed bacteria on chloramphenicol (12.5 μg/ml) LB medium.
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Incubate at 32°C for 36-48 hours
Insertion of Kan-SacB Cassette
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PCR amplify the Kan-SacB cassette with Phusion High Fidelity. Use 1-2 ng templates (the pDCE069 plasmid). 98°C 30 sec., (98°C 30 sec., 57°C 30 sec., 72°C 3.5 min.) for 40 cycles, 72°C 10 min. Gel-purify the PCR product, and elute in 30 μl ddH2O.
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Inoculate an overnight culture of SW102 cells containing the BAC. 5 ml LB + chloramphenicol. Incubate at 32°C.
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Next day, turn on two shakers: One at 32°C, the other at 42°C. Dilute 500 μl of the overnight culture in 25 ml LB with chloramphenicol (12.5 μg/ml), and incubate at 32°C to an OD600 of 0.6.
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Transfer 10 ml to another flask and heat-shock at 42°C for exactly 15 min. in a shaking water bath. The remaining culture is left at 32°C as the un-induced control.
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After 15 min., the two samples, induced and un-induced, are briefly cooled in an ice/waterbath slurry and then transferred to two 14 ml Falcon Round-Bottom tubes and pelleted using 4000 RPM at 0°C for 5 min.
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Pour off the supernatant and resuspend the pellet in 1 ml ice-cold ddH2O by gently swirling the tubes in the ice/waterbath slurry. No pipetting. When resuspended, add another 9 ml ice-cold ddH2O and pellet the samples again.
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Repeat step 10 twice.
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Remove all supernatant by inverting the tube on a paper towel. Gently resuspend the pellet as before to a final volume of 50 μl.
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Mix 25 μl of the freshly made electrocompetent cells with 200~300ng Kan-SacB fragment. Transfer the cocktail to a pre-cooled 0.1 cm cuvette.
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Transform by electroporation at 25 μF, 1.75 kV, and 200 ohms, twice.
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Transfer the bacteria to a tube with 1 ml LB medium. Incubate at 32°C for 1 h.
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Plate serial dilutions (100 μl, 100 μl of a 1:10 dilution, 100 μl 1:100) the transformed bacteria on chloramphenicol (12.5 μg/ml) and kanamycin (25 μg/ml) LB medium.
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Incubate at 32°C for 36-48 hours.
Replacement of Kan-SacB Cassette
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Inoculate an overnight culture of SW102 cells containing the BAC with Kan-SacB fragment. 5 ml LB + chloramphenicol. Incubate at 32°C.
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Next day, turn on two shakers: One at 32°C, the other at 42°C. Dilute 500 ?l of the overnight culture in 25 ml LB with chloramphenicol (12.5 μg/ml) and kanamycin (25 μg/ml), and incubate at 32°C to an OD600 of 0.6.
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Transfer 10 ml to another flask and heat-shock at 42°C for exactly 15 min. in a shaking water bath.
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After 15 min., the two samples, induced and un-induced, are briefly cooled in an ice/waterbath slurry and then transferred to two 14 ml Falcon Round-Bottom tubes and pelleted using 4000 RPM at 0°C for 5 min.
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Pour off the supernatant and resuspend the pellet in 1 ml ice-cold ddH2O by gently swirling the tubes in the ice/waterbath slurry. No pipetting. When resuspended, add another 9 ml ice-cold ddH2O and pellet the samples again.
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Repeat step 10 twice.
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Remove all supernatant by inverting the tube on a paper towel. Gently resuspend the pellet as before to a final volume of 50 μl.
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Mix 25 μl of the freshly made electrocompetent cells with 200~300ng DNA fragment (PCR product for marker insertion, and oligo for point mutation). Transfer the cocktail to a pre-cooled 0.1 cm cuvette.
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Transform by electroporation at 25 μF, 1.75 kV, and 200 ohms, twice.
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Transfer the bacteria to a tube with 1 ml Sucrose medium. Incubate at 32°C for 1 h.
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Plate serial dilutions (100 μl, 100 μl of a 1:10 dilution, 100 μl 1:100, and 100 μl 1:1000) the transformed bacteria on chloramphenicol (12.5 μg/ml) Sucrose medium.
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Incubate at 32°C for 36-48 hours.
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