DNA Isolation.docx

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University of Toronto St. George
John Coleman

DNA Isolation Introduction  Isolate DNA from fruit fly Drosophila melanogaster and understand the role of DNA and proteins Procedure 1. Obtain fruit flies that are already placed in a centrifuge tube. Place the flies on ice. 2. One student should use the regular Squishing Buffer (SB+) and the other should use Squishing buffer that does not contain either SDS/Proteinase K (SB-). 3. Each student should grind the flies to increase the surface area and allow the Squishing buffer to interact more. 4. A. Student should add SB+ (125 m) into tube and grind. Label tube. B. Student should add SB- (125 m) into tube and grind. Label tube. 5. Put tubes in 60 C water bath (Optimal temperature of the enzyme proteinase K 6. Centrifuge tubes for 3 mins at 14000 rpm. Balance the tubes by placing the tubes in opposite sides. 7. Top part should look oily and is the supernatant. There should be debris at the bottom. Using the P200 with the yellow tip transfer 200 m of the supernatant (contains the DNA) into a new test tube. 8. Add 20 m of 5M NaCl into the tube. 9. Add 436 m of ethanol, leave test tube in ice for 5 mins. 10.Centrifuge the tubes for 5 mins at 14000 rpm. Remove the ethanol by pouring in into a liquid waste bucket 11.Add 500 l of 70% ethanol to wash pellet. Centrifuge the tube for 5 mins at 14000 rpm. 12.Pour out ethanol and blot pellet 13.Add 50 l of TE to the tube and gently pipette the liquid up and d
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