Team:Fatih Turkey/Procedures

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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;">E.coli Compotent</h5>
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             <p>asdada</p><p>asdada</p><p>asdada</p><p>asdada</p><p>asdada</p><p>asdada</p><p>asdada</p><p>asdada</p>
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  <p>E.coli TOP10 Competent Cell</p>
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<p class="style1">MATERIALS</p>
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<p class="style1">·         Centrifuge</p>
 +
<p class="style1">·         Autoclave</p>
 +
<p class="style1">·         Incubator with shaker</p>
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<p class="style1">·         pH meter</p>
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<p class="style1">·         Stock competent cell</p>
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<p class="style1">·         LB broth</p>
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<p class="style1">·         Falcon</p>
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<p class="style1">·         Ice</p>
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<p class="style1">·         Liquid nitrogen</p>
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<p class="style1">·         Bacto yeast extract</p>
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<p class="style1">·         Bacto tryptone</p>
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<p class="style1">·         Magnesium sulfate</p>
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<p class="style1">·         Potassium hydroxide</p>
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<p class="style1">·         Potassium acetate</p>
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<p class="style1">·         Rubidium chloride</p>
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<p class="style1">·         Calcium chloride</p>
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<p class="style1">·         Manganese chloride</p>
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<p class="style1">·         Glycerol</p>
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<p class="style1">·         Dilute acetic acid</p>
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<p class="style1">·         Filter</p>
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<p class="style1">·         MOPS</p>
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<p class="style1">·         Dilute NAOH</p>
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<p class="style1">SOLUTIONS</p>
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<p class="style1"><br />
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  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>
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Revision as of 17:44, 27 October 2011

deneme baslik

Procedures

E.coli Compotent

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.

2011 © Fatih Medical School

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