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MIT7_13f08_lab22_Protocol_Transformation - MIT...

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MIT OpenCourseWare http://ocw.mit.edu 7.13 Experimental Microbial Genetics Fall 2008 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms .
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7 . 1 3 F a l l 2 0 0 8 P a g e | 1 Transforming DH5 α− T1 R Chemically Competent Cells Genotype DH5 α− T1 R : F- φ 80 lac Z Δ M15 Δ ( lac ZYA- arg F)U169 rec A1 end A1 hsd R17(rk-, mk+) pho A sup E44 thi -1 gyr A96 rel A1 ton A (confers resistance to phage T1) General Handling: Be extremely gentle when working with competent cells. Competent cells are highly sensitive to changes in temperature or mechanical lysis caused by pipetting. Transformation should be started immediately following the thawing of the cells on ice. Mix by swirling or tapping the tube gently, not by pipetting or vortexing. You will need the following items for transformation: • 37°C shaking and non-shaking incubator • 10 cm diameter LB agar plates with appropriate antibiotic • Ice bucket with ice • 42°C water bath Before Starting • Pour LB agar plates with appropriate antibiotic (this should be done WELL in advance of starting this protocol; see note below) • Equilibrate a water bath to 42°C • Warm the vial of SOC medium to room temperature • Spread X-Gal onto LB agar plates with antibiotic, if desired (bee section on blue white screening below) • Warm the plates in a 37°C incubator for 30 minutes • Obtain a test tube rack that will hold all transformation tubes so that they can all be put into a 42°C water bath at once.
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