Lab B Cell Fractionation(1).docx - Bio 122 Cells and Genetics Names Lab B – Cell Fractionation Goals 1 2 3 4 Use a blender and centrifuge to isolate

Lab B Cell Fractionation(1).docx - Bio 122 Cells and...

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Bio 122 Cells and Genetics Names: ______________________________ ______________________________ ______________________________ ______________________________ Lab B – Cell Fractionation Goals : 1. Use a blender and centrifuge to isolate cell fractions for the SDH assays. 2. Each student will be assessed in his/her competence with micropipettors. 3. Aliquot your mitochondrial and crude cell fractions for use over the next two weeks. 4. Begin to develop a hypothesis relating your assigned reagent to its effect on SDH activity. (Instructions for Hypothesis Assignment due Week 3, Lab C, below). Preparing for Lab : Review the protocol on cell fractionation Review the information on the proper use of a micropipettor Cell Fractionation Procedure: ALL REAGENTS MUST BE KEPT COLD THROUGHOUT PROCEDURES DO NOT DISCARD THE CENTRIFUGE TUBES!!! 1. Using scissors cut 12 g of thawed organ tissue into small pieces and place the pieces in the mortar. 2. Measure (100 mL) of ice-cold buffered sucrose using a graduated cylinder. 3. Add the buffered sucrose in 20 ml increments into the mortar and grind up the organ until all 40 ml of sucrose have been added. Grind the tissue slowly so you do not splash the mixture. 4. Add the minced tissue to the blender jar. 5. Add an additional 40 ml of fresh ice-cold sucrose to the blender jar. 6. Rinse the mortar with an additional 20 mls of sucrose buffer to collect any remaining tissue and add to the blender jar. 7. Blend the tissue/sucrose mixture in a blender until no lumps remain. 8. Decant (pour) the tissue/sucrose mixture through a double layer of cheesecloth placed over a funnel into a large ice-cold beaker. Rinse the blender with 20 ml fresh sucrose buffer and pour it through the cheesecloth and collect as above. This is a 10% (w/v) homogenate. Record the volume of the homogenate ________. 9. Take 12 ml of this crude homogenate, and place it into a clean 15 ml conical tube. Label as Crude Homogenate , and keep on ice. 10. Transfer an additional 40 ml of the 10% (w/v) homogenate from the beaker into an ice- cold centrifuge tube. 1
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Bio 122 Cells and Genetics 11. Place a piece of tape on the cap of the centrifuge tube and label it with section and group number. 12. Give the centrifuge tube to your TA. Your TA will centrifuge the 10% (w/v) homogenate for 10 minutes at 600 x g (2100 rpm) at 4°C. After the centrifugation is complete, the TA will pipet the resulting supernatant into a fresh, ice-cold centrifuge tube. The TA will centrifuge the supernatant for 20 minutes at 10,000 x g (9100 rpm) and then return the new tubes to the students.
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