Difference between revisions of "Team:Grenoble-Alpes/Protocols"

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<div id="electrophorese">
 
<div id="electrophorese">
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   <h2 class="protocol_name"></h2>
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   <h2 class="protocol_name">Electrophorese</h2>
 
   <h5>  
 
   <h5>  
−
      
+
  Materials
 +
  <li>1g Agarose (powder)</li>
 +
  <li>100mL TAE</li>
 +
  <li>Distilled Water</li>
 +
  <li>25µL Gel Red</li>
 +
  <br>
 +
  Methods
 +
  <ol>
 +
  <li>Gel preparation</li>
 +
     <ol>
 +
    <li>Dissolve 1g of agarose in 100mL of TAE (1X)</li>
 +
    <li>Pour the solution into the gel mould</li>
 +
    <li>Let the solution gel (almost 15 minutes)</li>
 +
    </ol>
 +
  <li>Preparation of the samples to deposit</li>
 +
  <li>The migration is done at 100V during 30 minutes</li>
 +
  <li>Revelation</li>
 +
    <ol>
 +
    <li>Prepare 250mL of distilled water in a tank</li>
 +
    <li>Add 25uL of Gel Red. Do not forget to use gloves to manipulate the Gel Red</li>
 +
    <li>Incubate the gel in the solution for 10 minutes at the obscurity</li>
 +
    <li>Wash the gel in a tank of distilled water</li>
 +
    </ol>   
 
   </h5>
 
   </h5>
 
   <a href="#exp" style="text-decoration: none;"><h2 class="cls">CLOSE</h2></a>
 
   <a href="#exp" style="text-decoration: none;"><h2 class="cls">CLOSE</h2></a>

Revision as of 13:17, 31 August 2017

Lab

Protocols

Preparations

LB Broth

Materials
  • 20g LB Broth Base (powder)
  • 1L Distillated Water

    • Methods
    1. Add 20g of LB Broth Base per 1L of distillated water
    2. Homogenize
    3. Autoclave at 121°C for 15 minutes

    CLOSE

    LB Agar

    Materials
  • 32g LB Agar (powder)
  • 1L Distilled Water

    • Methods
    1. Add 32g of LB Agar per 1L of distilled water
    2. Homogenize
    3. Autoclave at 121°C for 15 minutes

    CLOSE

    Chloramphenicol (Cam) 25ug/mL Stock

    Materials
  • 0,25g Chloramphenicol (25mg/mL)
  • 10mL Ethanol

    • Methods
    1. Add 0,25g of Cam per 10mL of ethanol
    2. Homogenize by vortexing
    3. Filter with a 22mm membrane filter
    4. Conserve at -20°C in 1mL aliquot

    CLOSE

    Petri Dish Preparation

    Materials
  • 20mL LB Agar
  • 20µL Cam (25µg/mL)

    • Methods
    1. Add of 20uL Cam in 20mL of liquid LB Agar
    2. Pour the solution in the plate
    3. Wait until the agar solidifies
    4. Conserve returned in 4°C

    CLOSE

    Glycerol 80%

    Materials
  • 80mL Glycerol 100%
  • 20mL Sterile Water

    • Methods
    1. Harvest 80mL of glycerol 100%
    2. Add 20mL of sterile water
    3. Conserve at 4°C

    CLOSE

    CaCl2 100mM

    Materials
  • 0,0555g CaCl2 (111g/mol)
  • 5mL Sterile Water

    • Methods
    1. Dissolve 0,056g of CaCl2 in 5mL of sterile water
    2. Conserve at 4°C

    CLOSE

    MgCl2 100mM

    Materials
  • 0,610g MgCl2 (203,31g/mol)
  • 30mL Sterile Water

    • Methods
    1. Dissolve 0,6105g of MgCl2 in 30mL of sterile water
    2. Conserve at 4°C

    CLOSE

    Sucrose 100mM

    Materials
  • 0,171g Sucrose
  • 5mL Sterile Water

    • Methods
    1. Dissolve 0,171g of sucrose in 5mL of sterile water
    2. Conserve at 4°C

    CLOSE

    IPTG 4mM

    Materials
  • 80µL IPTG (500mM)
  • 10mL LB
  • 10µL Cam (25ug/mL)

    • Methods
    1. Add 10µL of Cam in 10mL of LB
    2. Homogenize
    3. Remove 90µL of solution and add 80µL of IPTG
    4. Conserve at -20°C

    CLOSE

    Resuspension Solution for Lyophilized Bacteria

    Materials
  • 350µL DMSO
  • 1,5µL 2-mercaptoethanol 1%
  • 5mL Sterile Water

    • Methods
    1. Add 350µL of DMSO and 1,5µL of 2-mercaptoethanol in 5mL of sterile water
    2. Conserve at 4°C

    CLOSE

    Experimentations

    Electrophorese

    Materials
  • 1g Agarose (powder)
  • 100mL TAE
  • Distilled Water
  • 25µL Gel Red

    • Methods
    1. Gel preparation
      1. Dissolve 1g of agarose in 100mL of TAE (1X)
      2. Pour the solution into the gel mould
      3. Let the solution gel (almost 15 minutes)
    2. Preparation of the samples to deposit
    3. The migration is done at 100V during 30 minutes
    4. Revelation
      1. Prepare 250mL of distilled water in a tank
      2. Add 25uL of Gel Red. Do not forget to use gloves to manipulate the Gel Red
      3. Incubate the gel in the solution for 10 minutes at the obscurity
      4. Wash the gel in a tank of distilled water

    CLOSE

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