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

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<b>Demonstrate</b>
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<b>Freeze Drying Protocol</b>
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<h1>Demonstrate</h1>
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<h3>Gold Medal Criterion #4</h3>
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Teams that can show their system working under real world conditions are usually good at impressing the judges in iGEM. To achieve gold medal criterion #4, convince the judges that your project works. There are many ways in which your project working could be demonstrated, so there is more than one way to meet this requirement. This gold medal criterion was introduced in 2016, so check our what 2016 teams did to achieve their gold medals!
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Please see the <a href="https://2018.igem.org/Judging/Medals">2018 Medals Page</a> for more information.
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<li>Add 5 ml of sterile LB media and 5ul of chloramphenicol and ampicillin to a culture tube.</li>
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<li>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.</li>
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<li>Place culture tubes on a rack in the incubator. Set to shake and temperature to 36-38 C, and grow overnight.</li>
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<li>Centrifuge liquid culture and discard the supernatant. Resuspend the pellet in 5ml of Microbial Freeze Drying Buffer with tryptic soy broth.<li>
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<li>Aliquot 500ul of the suspension into sterile vials with stopper. </li>
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<li>Turn on the lyophilizer and start the condenser. Set the shelf to 4°C.</li>
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<li>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. <li>
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<li>Let the samples sit at -40°C for 1 hour to complete freezing. </li>
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<li>Turn on the vacuum pump.  Within 10-20 minutes, the vacuum should be under 200 millitorr (mtorr). </li>
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<li>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. </li>
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<li>For second drying, raise the shelf temperature to 20°C and dry for 2 hours.
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<li>With the stoppering mechanism, pit the stoppers on the vacuum. Turn off vacuum.
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<li>Store at 4°C in the dark.</li>
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Revision as of 22:27, 14 October 2018

CALM ELECTROPEN
BACKGROUND DATA & METHODS RESULTS DISCUSSION COLOR Q SUPPLEMENTARY REFERENCES
COLOR Q CHROME Q REFERENCES
PRINCIPLES & DESIGN MODEL & THEORY PRODUCT DESIGN REFERENCES
INTEGRATED HUMAN PRACTICES EDUCATION & ENGAGEMENT COLLABORATIONS
BASIC PARTS COMPOSITE PARTS
DESCRIPTION BACKGROUND DESIGN EXPERIMENTS LAB NOTEBOOK INTERLAB RESULTS DEMONSTRATE THE VISION
TEAM MEMBERS ATTRIBUTIONS

D E M O N S T R A T E



































Demonstrate


Freeze Drying Protocol


  1. Add 5 ml of sterile LB media and 5ul of chloramphenicol and ampicillin to a culture tube.
  2. 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.
  3. Place culture tubes on a rack in the incubator. Set to shake and temperature to 36-38 C, and grow overnight.
  4. Centrifuge liquid culture and discard the supernatant. Resuspend the pellet in 5ml of Microbial Freeze Drying Buffer with tryptic soy broth.
  6. Aliquot 500ul of the suspension into sterile vials with stopper.
  7. Turn on the lyophilizer and start the condenser. Set the shelf to 4°C.
  8. 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.
  10. Let the samples sit at -40°C for 1 hour to complete freezing.
  11. Turn on the vacuum pump. Within 10-20 minutes, the vacuum should be under 200 millitorr (mtorr).
  12. 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.
  13. For second drying, raise the shelf temperature to 20°C and dry for 2 hours.
  14. With the stoppering mechanism, pit the stoppers on the vacuum. Turn off vacuum.
  15. Store at 4°C in the dark.