"
Team:Alberta/Protocols
From 2012.igem.org
(Difference between revisions)
Rick24568509 (Talk | contribs) |
Rick24568509 (Talk | contribs) |
||
| Line 33: | Line 33: | ||
<li>Melt and pipette 25mL of LB agar into the Petri dish, and place the cap on top, allowing the magnet to rest in the molten agar. Let rest until solid and cool, then remove the cap and allow the surface to dry until it is free of excess moisture. | <li>Melt and pipette 25mL of LB agar into the Petri dish, and place the cap on top, allowing the magnet to rest in the molten agar. Let rest until solid and cool, then remove the cap and allow the surface to dry until it is free of excess moisture. | ||
<br> | <br> | ||
| - | + | ||
| + | |||
<font size=3> <b> | <font size=3> <b> | ||
Steps 4 and 5 may be completedin any desired order, depending on the approximate amount of time the substanceis meant to diffuse for. | Steps 4 and 5 may be completedin any desired order, depending on the approximate amount of time the substanceis meant to diffuse for. | ||
| Line 39: | Line 40: | ||
</font> | </font> | ||
<br> | <br> | ||
| - | + | ||
| + | |||
<li>Pipette 50µL of antibiotic (at a concentration high above the minimum inhibitory concentration) into the centre well. Be careful not to spill any. Immediately place the plate in a 37ºC incubator. | <li>Pipette 50µL of antibiotic (at a concentration high above the minimum inhibitory concentration) into the centre well. Be careful not to spill any. Immediately place the plate in a 37ºC incubator. | ||
<li>Evenly plate 200µL of cells with a resistance to the antibiotic over the flat surface of the plate. Immediately place the plate in a 37ºC incubator. | <li>Evenly plate 200µL of cells with a resistance to the antibiotic over the flat surface of the plate. Immediately place the plate in a 37ºC incubator. | ||
<li>Begin watching for results within 2 hours of plating. These will come in the form of a slight difference in texture between the zone in which cells are growing, and the zone in which they are not. It will be very subtle, and may need to be observed by shining light through the agar, or placing the plate on a black backdrop. As soon as it is observed, the radius of the zone must be measured from the edge of the well. The zone may expand. Continue recording the size and time until it stops changing. | <li>Begin watching for results within 2 hours of plating. These will come in the form of a slight difference in texture between the zone in which cells are growing, and the zone in which they are not. It will be very subtle, and may need to be observed by shining light through the agar, or placing the plate on a black backdrop. As soon as it is observed, the radius of the zone must be measured from the edge of the well. The zone may expand. Continue recording the size and time until it stops changing. | ||
</ol> | </ol> | ||
Revision as of 21:11, 24 August 2012
|
|
Protocols
Gradient plates
The following procedure is to be used to find valuesrelated to diffusion. All time values should be converted to seconds, from whenthe antibiotic was plated, and all distance values should be recorded incentimeters, from the edge of the well to the first sign of life at the edge ofthe “kill zone”. All materials should be kept as sterile as possible, barringthe damage of cells that are being plated.
- Draw a central cross on the cap of a Petri dish of radius 4.6cm
- Sterilize a tall cylindrical magnet of a height near, but not at the depth of the plate (a well that reaches the bottom of the plate will allow the antibiotic in question to seep under the agar rather than diffuse through it) and radius 0.25cm. Place the sterilized magnet on the inside of the Petri dish cap and, from outside the cap, adjust and secure the sterilized magnet with another magnet.
- Melt and pipette 25mL of LB agar into the Petri dish, and place the cap on top, allowing the magnet to rest in the molten agar. Let rest until solid and cool, then remove the cap and allow the surface to dry until it is free of excess moisture.
Steps 4 and 5 may be completedin any desired order, depending on the approximate amount of time the substanceis meant to diffuse for.
- Pipette 50µL of antibiotic (at a concentration high above the minimum inhibitory concentration) into the centre well. Be careful not to spill any. Immediately place the plate in a 37ºC incubator.
- Evenly plate 200µL of cells with a resistance to the antibiotic over the flat surface of the plate. Immediately place the plate in a 37ºC incubator.
- Begin watching for results within 2 hours of plating. These will come in the form of a slight difference in texture between the zone in which cells are growing, and the zone in which they are not. It will be very subtle, and may need to be observed by shining light through the agar, or placing the plate on a black backdrop. As soon as it is observed, the radius of the zone must be measured from the edge of the well. The zone may expand. Continue recording the size and time until it stops changing.
