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Transcript
DNA Transformation Lab E Coli with PCU Avery–MacLeod–McCarty experiment • Showed DNA is the substance that causes bacterial transformation Hershey- Chase • Showed DNA is the genetic material Propagating DNA in a host cell Requires a vector 1) Plasmid 2) Phage virus Plasmids Circular molecules of double stranded DNA that self replicating. They code for cell functions but are not essential Exchange of genes Prokaryotes exchange genes for diversity (asexual) 1) Transformation DNA is released into surrounding medium Recipient cells incorporate it into themselves from the medium 2) Transduction A phage virus attaches to bacterial cell and transfers its DNA into the bacterium Similar??? 3) Conjugation Plasmids travel between touching cells R factors Plasmids that contain resistance to antibiotics Transformation in streptococcus pneumoniae in mice Competent cells Cells that can be transformed by DNA Some can become competent by certain environmental conditions EX. E-coli E. Coli = Escherichia coli Normally harmless bacteria in your gut E. Coli can be artificially transformed by exposure to calcium chloride solution and thermally “shocked” To become receptive to foreign plasmid In this lab we will perform transformation of E Coli pUC8 = Ampicillin Resistant plasmid will be inserted into E coli LAB Sterile technique!! Don’t touch anything Lift Petri lid partially, pour/swab etc. Immediately reclose Open instruments and use immediately Do NOT touch working end Close quickly Incubation Incubate plates UPSIDE DOWN Antibiotic Ampicillin – Kills bacteria After mixing keep in freezer Thaw 30 mins. prior Needs Thermally shock to make E Coli competent to receive plasmid ICE BATH followed by Warm Water bath Bacteria Colonies Lawn – all together not separate 4 plates Luria Broth Luria Broth + AMP Broth AMP Broth + No antibiotic, no plasmid No antibiotic, With plasmid antibiotic, No plasmid antibiotic, With plasmid What do you expect Luria Broth - No antibiotic, no plasmid Normal growth = lawn of bacteria Luria Broth + No antibiotic, With plasmid Normal growth = lawn of bacteria AMP Broth - antibiotic, No plasmid NO growth, all killed AMP Broth + antibiotic, With plasmid Individual Colony growth of transformed resistant bacteria results Calculating efficiency of transformation Total mass of plasmid used Total mass = volume X concentration Volume = 10 µl Concentration = 1 µg/100µl = 0.1 µg plasmid Total volume of suspension Volume calcium .25 ml = chloride (chilled) 250µl calcium chloride Volume Plasmid 10 µl Plasmid Volume Luria broth .25 ml = 250 µl luria broth TOTAL Volume of Total = 510 µl suspension Fraction of suspension put on plate µl on plate/total volume .1 ml put on plate =100 µl Total volume = 510 µl (from above) 100 µl x 510 µl = 0.196 µl put on plate Total mass of plasmid in fraction (mass of plasmid x fraction on plate) Mass of plasmid = 0.1 µg Fraction on plate = 0.196 .1µg x 0.196 = Total mass of plasmid on plate = 0.0196 µg Number of colonies per µg of plasmid (# colonies counted/mass of plasmid put on plate) Count = # Mass of plasmid = 0.0196 µg Colonies per µg plasmid = …………