BISC314:Full Protocol: Difference between revisions
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For each of your isolates you should save a small amount of DNA for later identification. The protocol is below: <br> | For each of your isolates you should save a small amount of DNA for later identification. The protocol is below: <br> | ||
1. Flame the loop, allow it to cool for a few seconds and touch the cooled loop to a colony of your isolate, picking up a TINY bit of white growth from the bacterial colony. An invisible amount of growth obtained from just touching the cooled loop to the colony is fine.<br> | 1. Flame the loop, allow it to cool for a few seconds and touch the cooled loop to a colony of your isolate, picking up a TINY bit of white growth from the bacterial colony. An invisible amount of growth obtained from just touching the cooled loop to the colony is fine.<br> | ||
2. Place the loop within a 1.5 ml eppendorf tube containing 200 | 2. Place the loop within a 1.5 ml eppendorf tube containing 200 µl of water. <br> | ||
3. Vortex the tube to fully resuspend the isolate. <br> | 3. Vortex the tube to fully resuspend the isolate. <br> | ||
4. Freeze the tube at -20C for later. We will be identifying the most interesting isolates using molecular methods after our lab assays<br> | 4. Freeze the tube at -20C for later. We will be identifying the most interesting isolates using molecular methods after our lab assays<br> | ||
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<b> Interaction Assay </b> <br> | <b> Interaction Assay </b> <br> | ||
1. Create a 96-well template for your isolates. Using the excel template found here [[Media:template.xls]] make up the map for this 96-well plate - you should know where each of your 8 chosen isolates will be. <br> | 1. Create a 96-well template for your isolates. Using the excel template found here [[Media:template.xls]] make up the map for this 96-well plate - you should know where each of your 8 chosen isolates will be. <br> | ||
2. Using sterile technique, take 50 | 2. Using sterile technique, take 50 µl of your isolate from the overnight culture and place it into the appropriate well. Add another 100 µl of sterile media to the well to dilute the culture <br> | ||
3. When you are finished loading the top and side wells, use the p 20 micropipette to move 10 | 3. When you are finished loading the top and side wells, use the p 20 micropipette to move 10 µl from wells A2 - A8 into wells B2 - B8. Repeat this until H2 - H8 has 10 µl in it also. <br> | ||
4. Now we will add the organisms from wells B1 - H1 to the rest of the plate. Again, using the p 20, take 10 | 4. Now we will add the organisms from wells B1 - H1 to the rest of the plate. Again, using the p 20, take 10 µl from wells B1 - H1 and mix into the wells B2 - H2. Repeat this process until you have mixed all of the possible combinations of your isolates on this plate. <br> | ||
5. Each of your wells now has isolates growing by themselves and isolates mixed together. We will inoculate from this plate onto a square agar tray in order to observe what these organisms look like when grown together. For this we will use the multichannel micropipette. | 5. Each of your wells now has isolates growing by themselves and isolates mixed together. We will inoculate from this plate onto a square agar tray in order to observe what these organisms look like when grown together. For this we will use the multichannel micropipette. Using the multichannel, take 5 µl of your inocula and deposit a drop on top of the square agar tray. Repeat this until you have completed the full array. <br> | ||
6. Wait for your spots to dry before placing your trays upside down. <br> | |||
You should come in to the lab a few times this week to check on your assay and note any interesting results <br> | |||
'''LAB #4: Quorum sensing: chemical signaling within our community'''<br> | |||