Difference between revisions of "Team:Paris Bettencourt/Notebook/Riboswitch"

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<li>Recovery 2 hours then plate it on 3 different 100ug/mL Ampicillin LB</li>
 
<li>Recovery 2 hours then plate it on 3 different 100ug/mL Ampicillin LB</li>
 
<li> Incubate 37°C till tomorrow </li>
 
<li> Incubate 37°C till tomorrow </li>
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</ol>
  
 
<h2>Results</h2>
 
<h2>Results</h2>
 
The day after I had colonies growing, nothing on my control
 
The day after I had colonies growing, nothing on my control
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<li>
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<ul>
 
I took 2 different colonies and do a 8 hours culture in two different tubes T1 an T2 in LB + Amp 100ug/mL, in order to do a glycerol stock.
 
I took 2 different colonies and do a 8 hours culture in two different tubes T1 an T2 in LB + Amp 100ug/mL, in order to do a glycerol stock.
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</li>
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</ul>
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<br><br>
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<h1 class="date one">July 29th</h1>
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<h2>Goal</h2>
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Making chemically competent cells
 +
 
 +
<h2>Procedure</h2>
 +
 
 +
I followed the following method 
 +
<li>The day before, start an overnight Liquid Culture of the E. coli strain. (5mL of LB + Antibiotics + Single colony)</li>
 +
<li>The next day, set the Centrifuge at 4°C and place on ice a tube containing 15mL of 100mM CaCl2 and a tube containing 4mL of 100mM CaCl2 + 15% Glycerol.</li>
 +
<li>In an Erlenmeyer, add 50mL of LB and 250uL of the Liquid Culture, (for quicker growth, use a larger Erlenmeyer)</li>
 +
<li>Put the Erlenmeyer to shake at 37°C until the OD(600) reaches 0.4 - 0.5.</li>
 +
<li>When the OD(600) is reached, transfer in a 50mL Falcon tube and centrifuge 10min at 4°C.</li>
 +
<li>Remove the supernatant and resuspend in 15mL of ice cold 100mM CaCl2. -Leave on ice for 4h.</li>
 +
<li>Centrifuge 10min at 4°C.</li>
 +
<li>Remove the supernatant and resuspend in 4mL of ice cold 100mM CaCl2 + 15% Glycerol.</li>
 +
<li>Aliquot in 1.5mL eppendorf tubes and store at -80°C until use.</li>
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</ol>

Revision as of 16:45, 1 September 2015

July 27th

Goal

E coli DH5-alpha transformation with RFP from iGEM plate 4 - 2H (pSB1A3 backbone)

Procedure

  1. I took iGEM plate 4 - 2H (RFP, BBa_J04450 in PSB1A3 backbone) and resuspend it in 10 uL of distilled water.
  2. Used E coli competent cells from NEB 2 tubes c2987 (50uL) : 1 control (C) and one for transformation (T)
  3. Introduced 2 uL (200-300pg/ul) of my suspended plasmid solution in T.
  4. Waiting 30 min in ice
  5. Heat shock 30 sec 42°C
  6. Then 5min in ice
  7. Add 200uL SOC medium
  8. Recovery 2 hours then plate it on 3 different 100ug/mL Ampicillin LB
  9. Incubate 37°C till tomorrow

Results

The day after I had colonies growing, nothing on my control
    I took 2 different colonies and do a 8 hours culture in two different tubes T1 an T2 in LB + Amp 100ug/mL, in order to do a glycerol stock.


July 29th

Goal

Making chemically competent cells

Procedure

I followed the following method
  • The day before, start an overnight Liquid Culture of the E. coli strain. (5mL of LB + Antibiotics + Single colony)
  • The next day, set the Centrifuge at 4°C and place on ice a tube containing 15mL of 100mM CaCl2 and a tube containing 4mL of 100mM CaCl2 + 15% Glycerol.
  • In an Erlenmeyer, add 50mL of LB and 250uL of the Liquid Culture, (for quicker growth, use a larger Erlenmeyer)
  • Put the Erlenmeyer to shake at 37°C until the OD(600) reaches 0.4 - 0.5.
  • When the OD(600) is reached, transfer in a 50mL Falcon tube and centrifuge 10min at 4°C.
  • Remove the supernatant and resuspend in 15mL of ice cold 100mM CaCl2. -Leave on ice for 4h.
  • Centrifuge 10min at 4°C.
  • Remove the supernatant and resuspend in 4mL of ice cold 100mM CaCl2 + 15% Glycerol.
  • Aliquot in 1.5mL eppendorf tubes and store at -80°C until use.