Difference between revisions of "Team:Lambert GA/Demonstrate"

Revision as of 22:32, 14 October 2018

D E M O N S T R A T E

Demonstrate

Freeze Drying Protocol

Add 5 ml of sterile LB media and 5ul of chloramphenicol and ampicillin to a culture tube.
Obtain the desired plate. Take 1-3 colonies from the biosensor cells’ plate with a sterilized inoculating loop. Stir vigorously to ensure colonies are dispensed into solution.
Place culture tubes on a rack in the incubator. Set to shake and temperature to 36-38 C, and grow overnight.
Centrifuge liquid culture and discard the supernatant. Resuspend the pellet in 5ml of Microbial Freeze Drying Buffer with tryptic soy broth.
Aliquot 500ul of the suspension into sterile vials with stopper.
Turn on the lyophilizer and start the condenser. Set the shelf to 4°C.
Center the vials on the shelf. Either manually or with programmed controls, freeze the samples down to -40°C. This should take about 30-60 minutes, but it is very dependent upon the lyophilizer. If the rate of freezing can be controlled, a practical rate is to drop the temperature by 1°C per minute. The samples should be visually frozen.
Let the samples sit at -40°C for 1 hour to complete freezing.
Turn on the vacuum pump. Within 10-20 minutes, the vacuum should be under 200 millitorr (mtorr).
After the vacuum is below 200 mtorr, increase the temperature of the shelf for primary drying. The temperature can be up to -15°C. Let continue overnight.
For second drying, raise the shelf temperature to 20°C and dry for 2 hours.
With the stoppering mechanism, pit the stoppers on the vacuum. Turn off vacuum.
Store at 4°C in the dark.

In-Field Preparation of Cells

*Tubes should be on ice at all times unless centrifuging*

Resuspend in 5 ml of LB agar. Incubate for two hour.
Aliquot 1 ml of that liquid culture into 4 ml of sterile LB.
Shake in an incubator overnight. If no incubator present, let sit at room temperature for a day and a half.
Add the 5 ml liquid culture into 500 ml of sterile LB.
Using the portable spectrophotometer, check the optical density every hour. Continue growing until the OD is 0.6.
Pour cells into 50 ml conical tubes, on ice. Keep there for 30 minutes.
Pipet up the bottom 1.5 ml in the tube and transfer to a microcentrifuge tube.
With the 3D-fuge, centrifuge cells for 20 minutes, discard supernatant.
Add 1.5 ml of ice-cold sterile into the microcentrifuge to resuspend. Add into empty 50 ml conical tube.
Add another 11.5 ml of ice-cold sterile milliQ water to each conical tube, pipet up and down.
Combine samples into 2 total conical tubes.
Let sit on ice for 30 minutes. Take bottom 1.5 ml of each sample and transfer into a empty microcentrifuge tube.
With 3D-fuge, centrifuge for 20 minutes, discard supernatant, and repeat steps 9 and 10.
Let sit for 30 minutes on ice.
Transfer bottom 1.5 ml into a microcentrifuge tube. Centrifuge for 20 minutes, discard supernatant, and add 1.5 ml of ice-cold 10% glycerol.
Transfer the microcentrifuge into an empty conical tube. Add 23.5 ml of ice-cold 10% glycerol.
Let sit for 30 minutes on ice. Repeat steps 15 and 16.
Let sit for 30 minutes on ice. Take bottom 1.5 ml and transfer into a microcentrifuge tube. Centrifuge with 3D-fuge for 20 minutes.
Resuspend in 1.5 ml of ice-cold 10% glycerol. Transfer into conical tube and add 2.5 ml of ice-cold 10% glycerol.
Aliquot 1 ml into microcentrifuge tubes. Store in -80 freezer.

@@ Line 291: / Line 291: @@
 <li>Store at 4°C in the dark.</li>
 </ol>
+<div id="target3"></div>
+<div id="subheading2">
+<b>In-Field Preparation of Cells</b>
+</div>
+<br><br>
+<div id="content2">
+<i style="font-size:20px;">*Tubes should be on ice at all times unless centrifuging*</i>
+<ol>
+<li>Resuspend in 5 ml of LB agar. Incubate for two hour. </li>
+<li>Aliquot 1 ml of that liquid culture into 4 ml of sterile LB.</li>
+<li>Shake in an incubator overnight. If no incubator present, let sit at room temperature for a day and a half.</li>
+<li>Add the 5 ml liquid culture into 500 ml of sterile LB.</li>
+<li>Using the portable spectrophotometer, check the optical density every hour. Continue growing until the OD is 0.6.</li>
+<li>Pour cells into 50 ml conical tubes, on ice. Keep there for 30 minutes.</li>
+<li>Pipet up the bottom 1.5 ml in the tube and transfer to a microcentrifuge tube.</li>
+<li>With the 3D-fuge, centrifuge cells for 20 minutes, discard supernatant.</li>
+<li>Add 1.5 ml of ice-cold sterile into the microcentrifuge to resuspend. Add into empty 50 ml conical tube.</li>
+<li>Add another 11.5 ml of ice-cold sterile milliQ water to each conical tube, pipet up and down.</li>
+<li>Combine samples into 2 total conical tubes.</li>
+<li>Let sit on ice for 30 minutes. Take bottom 1.5 ml of each sample and transfer into a empty microcentrifuge tube.</li>
+<li>With 3D-fuge, centrifuge for 20 minutes, discard supernatant, and repeat steps 9 and 10.</li>
+<li>Let sit for 30 minutes on ice. </li>
+<li>Transfer bottom 1.5 ml into a microcentrifuge tube. Centrifuge for 20 minutes, discard supernatant, and add 1.5 ml of ice-cold 10% glycerol.</li>
+<li>Transfer the microcentrifuge into an empty conical tube. Add 23.5 ml of ice-cold 10% glycerol.</li>
+<li>Let sit for 30 minutes on ice. Repeat steps 15 and 16. </li>
+<li>Let sit for 30 minutes on ice. Take bottom 1.5 ml and transfer into a microcentrifuge tube. Centrifuge with 3D-fuge for 20 minutes. </li>
+<li>Resuspend in 1.5 ml of ice-cold 10% glycerol. Transfer into conical tube and add 2.5 ml of ice-cold 10% glycerol.</li>
+<li>Aliquot 1 ml into microcentrifuge tubes. Store in -80 freezer.</li>
+</ol>
 </div>