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#Go to the instructor's computer in the front of the lab and record your TASTER phenotype as T (taster), NT (non-taster), or PT (partial taster). You can choose any of the unique identifier numbers that are assigned to your lab section. Write down in your lab notebook the number you have chosen so you don't forget it! | #Go to the instructor's computer in the front of the lab and record your TASTER phenotype as T (taster), NT (non-taster), or PT (partial taster). You can choose any of the unique identifier numbers that are assigned to your lab section. Write down in your lab notebook the number you have chosen so you don't forget it! | ||
#Use a permanent marker to label the top of a 1.5-mL microfuge tube with your number, a paper cup and also label your centrifuge tube containing your saliva with your assigned number. | #Use a permanent marker to label the top of a 1.5-mL microfuge tube with your number, a paper cup and also label your centrifuge tube containing your saliva with your assigned number. | ||
#Centrifuge the saliva | #Centrifuge the saliva in the desktop centrifuges for 5 minutes to pellet your cells. | ||
#IF your cells have made a tight pellet on the bottom of the tube, turn the tube upside down over your waste beaker to decant the supernatant, being careful to avoid dislodging the cell pellet at the bottom. Your instructor will demo how to do this. Do not return the tube to upright! Instead bring a paper towel over to the tube that is draining into the waste beaker and continue draining the tube onto the paper towel for a few minutes. Keep an eye on the pellet and make sure that it does not slide out! | #IF your cells have made a tight pellet on the bottom of the tube, turn the tube upside down over your waste beaker to decant the supernatant, being careful to avoid dislodging the cell pellet at the bottom. Your instructor will demo how to do this. Do not return the tube to upright! Instead bring a paper towel over to the tube that is draining into the waste beaker and continue draining the tube onto the paper towel for a few minutes. Keep an eye on the pellet and make sure that it does not slide out! | ||
#Resuspend ''all'' of your cells in 400 μL of PBS (phosphate-buffered saline) by vortexing at highest speed. You may need to use a micropipet tip to dislodge some of the cells from the tube wall. Make sure that all cells are completely resuspended before proceeding to the next step. | #Resuspend ''all'' of your cells in 400 μL of PBS (phosphate-buffered saline) by vortexing at highest speed. You may need to use a micropipet tip to dislodge some of the cells from the tube wall. Make sure that all cells are completely resuspended before proceeding to the next step. | ||
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#Add 400 μL of 100% ethanol to the microfuge tube. Mix thoroughly by vortexing for 15 seconds. | #Add 400 μL of 100% ethanol to the microfuge tube. Mix thoroughly by vortexing for 15 seconds. | ||
#Briefly microfuge to prevent your solution from sticking to the inside of the lid. (Press the short spin button on your microfuge.) | #Briefly microfuge to prevent your solution from sticking to the inside of the lid. (Press the short spin button on your microfuge.) | ||
#Locate a DNeasy spin column and three collection tubes. Remove 700 μL of your sample and apply to the DNeasy spin column (while the column is in the collection tubes). Microfuge at 8000 rpm for 1 minute. '''These spin columns will only hold about 700 μL of fluid, so you will repeat this process in step | #Locate a DNeasy spin column and three collection tubes. Remove 700 μL of your sample and apply to the DNeasy spin column (while the column is in the collection tubes). Microfuge at 8000 rpm for 1 minute. '''These spin columns will only hold about 700 μL of fluid, so you will repeat this process in step 14 in order to move ''all'' of your DNA onto a single spin column.''' | ||
#Remove the spin column and discard the flow-through by dumping the contents of the collection tube into your liquid waste container. | #Remove the spin column and discard the flow-through by dumping the contents of the collection tube into your liquid waste container. | ||
#Place the spin column back into the same collection tube (which should now be empty). Add the remainder of your sample to the spin column. Microfuge at 8000 rpm for 1 minute. '''If you have any precipitate by this stage in your original tube, pipette all of this (along with the liquid) onto the spin column. The DNA from your cells is now binding to the silica-gel-membrane in the spin column.''' | #Place the spin column back into the same collection tube (which should now be empty). Add the remainder of your sample to the spin column. Microfuge at 8000 rpm for 1 minute. '''If you have any precipitate by this stage in your original tube, pipette all of this (along with the liquid) onto the spin column. The DNA from your cells is now binding to the silica-gel-membrane in the spin column.''' |
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