Team:Fatih Turkey/Procedures

From 2011.igem.org

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<a href="https://2011.igem.org/Team:Fatih_Turkey/Human_Practice">Human Practice</a>
<a href="https://2011.igem.org/Team:Fatih_Turkey/Human_Practice">Human Practice</a>
<ul>
<ul>
-
<li><a href="https://2011.igem.org/Team:Fatih_Turkey/Sporocide">Sporocide</a></li>
+
<li><a href="https://2011.igem.org/Team:Fatih_Turkey/Sporocide">Sporicide</a></li>
<li><a href="https://2011.igem.org/Team:Fatih_Turkey/iGEM_for_7_to_77">iGEM for 7 to 77</a></li>
<li><a href="https://2011.igem.org/Team:Fatih_Turkey/iGEM_for_7_to_77">iGEM for 7 to 77</a></li>
<li><a href="https://2011.igem.org/Team:Fatih_Turkey/game">Game</a></li>
<li><a href="https://2011.igem.org/Team:Fatih_Turkey/game">Game</a></li>
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                                                                                 <li><a
                                                                                 <li><a
href="https://2011.igem.org/Team:Fatih_Turkey/Safety">Safety</a></li>
href="https://2011.igem.org/Team:Fatih_Turkey/Safety">Safety</a></li>
 +
<li><a href="https://2011.igem.org/Team:Fatih_Turkey/collaboration">Collaboration</a></li>
</ul>
</ul>
</li>
</li>
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<div class="bar-title-whole">
<div class="bar-title-whole">
  <div class="bar-title">
  <div class="bar-title">
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      <h2 style="font-family: Verdana, Arial, SunSans-Regular, sans-serif;">Lab Garage</h2>
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      <h2 style="font-family: Verdana, Arial, SunSans-Regular, sans-serif;">Procedures</h2>
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      <h5 style="font-family: Verdana, Arial, SunSans-Regular, sans-serif;">Procedures</h5>
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      <h5 style="font-family: Verdana, Arial, SunSans-Regular, sans-serif;">E.coli Compotent</h5>
    </div>
    </div>
  <div class="bar-title-sh-left"></div>
  <div class="bar-title-sh-left"></div>
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         <div style="float: left; width: 220px; border-radius: 10px; position: absolute; top: 50px; left: 0; background: #EEE; border: solid 3px #666;">
         <div style="float: left; width: 220px; border-radius: 10px; position: absolute; top: 50px; left: 0; background: #EEE; border: solid 3px #666;">
             <ol>
             <ol>
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               <li type="A"><a href="https://2011.igem.org/Team:Fatih_Turkey/ecolicompetent">E.coli Competent preperation</a>               
+
               <li type="A"><a href="https://2011.igem.org/Team:Fatih_Turkey/ecolicompetent">E.coli Competent</a>               
               </li>
               </li>
               <li type="A"><a href="https://2011.igem.org/Team:Fatih_Turkey/ecolitransformation">E.coli Trasformation</a></li>
               <li type="A"><a href="https://2011.igem.org/Team:Fatih_Turkey/ecolitransformation">E.coli Trasformation</a></li>
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               <li type="A"><a href="https://2011.igem.org/Team:Fatih_Turkey/bsubtilis">B.subtilis transformation and competent</a></li>
+
               <li type="A"><a href="https://2011.igem.org/Team:Fatih_Turkey/bsubtilis">B.subtilis transformation</a></li>
 +
            <li type="A"><a href="https://2011.igem.org/Team:Fatih_Turkey/sporicideprocedure">Sporicide</a></li>
             </ol>
             </ol>
         </div>
         </div>
         <div style="width: 654px; border: solid 3px #666; border-radius: 10px; padding-left: 80px; margin-left: 170px;">
         <div style="width: 654px; border: solid 3px #666; border-radius: 10px; padding-left: 80px; margin-left: 170px;">
-
             <p>asdada</p><p>asdada</p><p>asdada</p><p>asdada</p><p>asdada</p><p>asdada</p><p>asdada</p><p>asdada</p>
+
             <div>
 +
<div class="style1">
 +
  <p>E.coli TOP10 Competent Cell</p>
 +
</div>
 +
<p class="style1">MATERIALS</p>
 +
<p class="style1">·         Centrifuge</p>
 +
<p class="style1">·         Autoclave</p>
 +
<p class="style1">·         Incubator with shaker</p>
 +
<p class="style1">·         pH meter</p>
 +
<p class="style1">·         Stock competent cell</p>
 +
<p class="style1">·         LB broth</p>
 +
<p class="style1">·         Falcon</p>
 +
<p class="style1">·         Ice</p>
 +
<p class="style1">·         Liquid nitrogen</p>
 +
<p class="style1">·         Bacto yeast extract</p>
 +
<p class="style1">·         Bacto tryptone</p>
 +
<p class="style1">·         Magnesium sulfate</p>
 +
<p class="style1">·         Potassium hydroxide</p>
 +
<p class="style1">·         Potassium acetate</p>
 +
<p class="style1">·         Rubidium chloride</p>
 +
<p class="style1">·         Calcium chloride</p>
 +
<p class="style1">·         Manganese chloride</p>
 +
<p class="style1">·         Glycerol</p>
 +
<p class="style1">·         Dilute acetic acid</p>
 +
<p class="style1">·         Filter</p>
 +
<p class="style1">·         MOPS</p>
 +
<p class="style1">·         Dilute NAOH</p>
 +
<p class="style1">SOLUTIONS</p>
 +
<p class="style1"><br />
 +
  Preparation of Psi broth (per liter)<br />
 +
  - 5 g bacto yeast extract<br />
 +
  - 20 g bacto tryptone<br />
 +
  - 5 g magnesium sulfate<br />
 +
  - PH 7.6 with potassium hydroxide <br />
 +
  - Autoclave 40 min<br />
 +
  <br />
 +
  Preparation of TfbI (per 200 ml)<br />
 +
  - 0.588 g potassium acetate (final molarity/conc= 30 mM)<br />
 +
  - 2.42 g rubidium chloride (final molarity/conc= 100 mM)<br />
 +
  - 0.294 g calcium chloride (final molarity/conc= 10 mM)<br />
 +
  - 2.0 g manganese chloride (final molarity/conc= 50 mM)<br />
 +
  - 30 mL glycerol (15% v/v)<br />
 +
  - Adjust PH 5.8 with dilute acetic acid<br />
 +
  - Sterilize with filter <br />
 +
  <br />
 +
  Preparation of TfbII (per 100 ml)<br />
 +
  - 0.21 g MOPS (final molarity/conc= 10 mM)<br />
 +
  - 1.1 g calcium chloride (final molarity/conc= 75 mM)<br />
 +
  - 0.121 g rubidium chloride (final molarity/conc= 10 mM)<br />
 +
  - 15 mL glycerol (15% v/v)<br />
 +
  <strong>- </strong>Adjust PH 6.5 with dilute NaOH <br />
 +
  <strong>- </strong>Sterilize with filter</p>
 +
<p class="style1">- Inoculate streak plates from liquid stock competent cells and incubate overnight at 37˚C </p>
 +
<p class="style1">- Put 10 mL LB + colony  into 50ml falcon. Incubate overnight at 37˚C with shaker</p>
 +
<p class="style1">- Inoculate 200 ul to 1000 ul from overnight culture into 100-500 ml Psi broth  (scale up or down as needed). Incubate at 37 C with aeration to A600=0.6-0.7 </p>
 +
<p class="style1">- Ice 15 min.  From this step onward the cells must remain <strong>COLD.</strong> (4C or on ice)<br />
 +
  - Pellet cells in appropriate centrifuge tube 3-5000 x g 5 min (~5000 rpm in a Sorvall SS-34 rotor)<br />
 +
  - Discard supernatant and add 0.4 volume (ie of original volume, here it is 40-400 ml) TfbI, resuspend and ice 15 min.<br />
 +
  - Pellet cells in appropriate centrifuge tube 3-5000 x g 5 min (~5000 rpm in a Sorvall SS-34 rotor)<br />
 +
  - Discard supernatant and resuspend in 0.04 volume TfbII, ice 15 min and either use immediately or quick freeze at -70C for storage. I usually save these in 100ul  to 200ul aliquots. Quick freeze in ethanol-dry ice or liquid nitrogen prior to storage in a -70 to -80 C freezer. Thaw on ice just before using in a transformation experiment.</p>
         </div>
         </div>
     </div>
     </div>

Latest revision as of 19:51, 28 October 2011

deneme baslik

E.coli TOP10 Competent Cell

MATERIALS

·         Centrifuge

·         Autoclave

·         Incubator with shaker

·         pH meter

·         Stock competent cell

·         LB broth

·         Falcon

·         Ice

·         Liquid nitrogen

·         Bacto yeast extract

·         Bacto tryptone

·         Magnesium sulfate

·         Potassium hydroxide

·         Potassium acetate

·         Rubidium chloride

·         Calcium chloride

·         Manganese chloride

·         Glycerol

·         Dilute acetic acid

·         Filter

·         MOPS

·         Dilute NAOH

SOLUTIONS


Preparation of Psi broth (per liter)
- 5 g bacto yeast extract
- 20 g bacto tryptone
- 5 g magnesium sulfate
- PH 7.6 with potassium hydroxide 
- Autoclave 40 min

Preparation of TfbI (per 200 ml)
- 0.588 g potassium acetate (final molarity/conc= 30 mM)
- 2.42 g rubidium chloride (final molarity/conc= 100 mM)
- 0.294 g calcium chloride (final molarity/conc= 10 mM)
- 2.0 g manganese chloride (final molarity/conc= 50 mM)
- 30 mL glycerol (15% v/v)
- Adjust PH 5.8 with dilute acetic acid
- Sterilize with filter 

Preparation of TfbII (per 100 ml)
- 0.21 g MOPS (final molarity/conc= 10 mM)
- 1.1 g calcium chloride (final molarity/conc= 75 mM)
- 0.121 g rubidium chloride (final molarity/conc= 10 mM)
- 15 mL glycerol (15% v/v)
Adjust PH 6.5 with dilute NaOH 
Sterilize with filter

- Inoculate streak plates from liquid stock competent cells and incubate overnight at 37˚C

- Put 10 mL LB + colony  into 50ml falcon. Incubate overnight at 37˚C with shaker

- Inoculate 200 ul to 1000 ul from overnight culture into 100-500 ml Psi broth  (scale up or down as needed). Incubate at 37 C with aeration to A600=0.6-0.7

- Ice 15 min.  From this step onward the cells must remain COLD. (4C or on ice)
- Pellet cells in appropriate centrifuge tube 3-5000 x g 5 min (~5000 rpm in a Sorvall SS-34 rotor)
- Discard supernatant and add 0.4 volume (ie of original volume, here it is 40-400 ml) TfbI, resuspend and ice 15 min.
- Pellet cells in appropriate centrifuge tube 3-5000 x g 5 min (~5000 rpm in a Sorvall SS-34 rotor)
- Discard supernatant and resuspend in 0.04 volume TfbII, ice 15 min and either use immediately or quick freeze at -70C for storage. I usually save these in 100ul  to 200ul aliquots. Quick freeze in ethanol-dry ice or liquid nitrogen prior to storage in a -70 to -80 C freezer. Thaw on ice just before using in a transformation experiment.