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NAU BIO 205L - BIO205_Prelab10

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Kristina SimonovicSection O30 March 2016Exercise TenTitle:Bacterial TransformationHypothesis:If there is growth of colonies only in the spots on the plate with the recipient with no growth on the control plate then transformation has occurred.Methods:Add 1.5 mL of the wild-type nutrient broth culture to the microcentrifuge tube and pellet the cells. Pour off the supernatant and resuspend the pellet in 1.5 mL of lysis solution. Use a steri-loop to detach the cell pellet from the bottom before vortexing. Incubate at 60 degree C until the turbidity is gone and the solution is clear. Cool to room temperature. Aseptically prepare 1/10 and 1/100 dilutions of the lysate in sterile water. Label one of the plates with a T for transformation and the other with a C for control. On the back of each plate draw three dime sized circles in a triangular pattern and label the circles U for undiluted, 1/10 and 1/100. Spread the plate labeled T with 0.1 mL of the tryptophan-requiring mutant and allow the plate the dry. Do not spread the control plate. Use one 10 microliters steri-loop for each of the DNA solutions that you have prepared. Place 10 microliters of DNA from each solution onto the control plate in the areas indicated. You must apply the DNA to the control plate first. Repeat the process with the plate marked T. Allow the spots to dry and handle the plates gently so that the drops do not run together. Incubate at 37 degrees C for 48 hours. Growth of colonies only in the spots on the plate with the recipient with no growth on the control plate indicates that transformation has occurred.Safety Concerns:1. Carefully prepare dilutions of 1/10 and 1/1002. Use a control plate to differentiate growth colonies3. Use the aseptic technique to ensure the dilutions are pureRelevance:By conducting this experiment, one will be able to confirm the way in which DNA is transferred for this specific

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