3,811
edits
BISC209/F13: Lab7: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
→Antagonistic and Mutualistic Interactions Analysis
| Line 129: | Line 129: | ||
[[Image:Interactions_slide5.jpg]] <BR> | [[Image:Interactions_slide5.jpg]] <BR> | ||
<BR> | <BR> | ||
5. Use an 8 channel multichannel pipet with the first tip removed (you are not yet transferring from well A1) to transfer 10 μl of the contents of the 7 wells in the top row: A2 (containing isolate #2 etc) through A8 to the corresponding empty wells in each row as indicated by the yellow arrows. Change tips between each transfer from row 1 to row 2, then row 1 to row 3, etc. until each row indicated by the yellow arrows has been inoculated with 10 µL from the corresponding well in row 1. <BR> | |||
<BR> | <BR> | ||
If you are using the multichannel pipette, be sure that you work slowly and check that each pipette tip is evenly filled. You may need to tighten the tips by hand, if so be sure to only touch the part of the tip that sits on the multichannel pipette, you wouldn't want to contaminate your wells with human organsisms!<BR><BR> | |||
[[Image:Interactions_slide6.jpg]] <BR> | [[Image:Interactions_slide6.jpg]] <BR> | ||
<BR> | <BR> | ||
6. Using all 8 channels of a multichannel pipet, | 6. Using all 8 channels of a multichannel pipet, transfer 10 μl of the contents of column 1 (all 8 wells) to each of the corresponding wells in the other columns. Again, change tips between each transfer: A1 through H1, to A2 to H2 then A1 through H1 to A3 to H3 etc. until each of the columns indicated by the red arrows has been inoculated with 10 µL from the corresponding well in column 1. <BR> | ||
7. Mix the contents of the well by moving the plate in gentle circles.<BR> | 7. Mix the contents of the well by moving the plate in gentle circles.<BR> | ||
<BR> | <BR> | ||
8. NEXT, we will inoculate a labeled square (NUNC) tray containing nutrient agar medium with about 5 μl of the contents of the wells we just prepared. For this step we will use either a tool called a "frogger" or a multichannel micropipette. If using the frogger, dip the frogger tips into 96 well plate to attract a drop of inoculum onto the end of each steel tip. Be sure the frogger is actually sitting in the liquid of all 64 wells. Transfer the Frogger to the surface of the sterile NA in the square NUNC plate. Do not break the surface of the agar but make sure your pressure is even so every steel tip has touched the agar surface and deposited the same inoculum. Be sure to disinfect the frogger by dipping it into a series of disinfectant and rinse solutions that you will find at the '''cleaning station''' prepared for you . <BR><BR> | 8. NEXT, we will inoculate a labeled square (NUNC) tray containing nutrient agar medium with about 5 μl of the contents of the wells we just prepared. For this step we will use either a tool called a "frogger" or a multichannel micropipette. If using the frogger, dip the frogger tips into 96 well plate to attract a drop of inoculum onto the end of each steel tip. Be sure the frogger is actually sitting in the liquid of all 64 wells. Transfer the Frogger to the surface of the sterile NA in the square NUNC plate. Do not break the surface of the agar but make sure your pressure is even so every steel tip has touched the agar surface and deposited the same inoculum. < BR> | ||
Be sure to disinfect the frogger by dipping it into a series of disinfectant and rinse solutions that you will find at the '''cleaning station''' prepared for you . <BR><BR> | |||
9. If the frogger is not available, use an 8 channel multichannel pipet set to 5µl and remove 5μL of culture from each well of your culture dish and deposit all of it onto an area of the NA square agar NUNC plate that is in the same location as in the 96 well culture dish. Again, be sure the tips are on tightly before loading the pipet. Repeat this procedure, with new tips, for each ROW of 8 wells until you have completed depositing the full array in the same orientation as the 48 wells.<br><BR> | 9. If the frogger is not available, use an 8 channel multichannel pipet set to 5µl and remove 5μL of culture from each well of your culture dish and deposit all of it onto an area of the NA square agar NUNC plate that is in the same location as in the 96 well culture dish. Again, be sure the tips are on tightly before loading the pipet. Repeat this procedure, with new tips, for each ROW of 8 wells until you have completed depositing the full array in the same orientation as the 48 wells.<br><BR> | ||
