Team:Bielefeld-Germany/Labjournal/Molecular

From 2013.igem.org

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<h1>Protocols</h1>
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<h1 style="color:#ff6600;"> Genetic engineering protocols</h1>
 
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<br>
 
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<!--<h2>Standard Protocols</h2> -->
 
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<div class="iso_whole_headline">
 
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<a name="iso_whole"><span style="color:#ff6600">[Whole Genome Isolation]</span></a>
 
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<br>
 
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For the isolation following kit has been used:
 
-
Fermentas GeneJET™ Plasmid Miniprep Kit
 
-
<p> - Centrifuge 10 mL of over-night liquid culture</p>
 
-
<p> - Resuspend pellet in 800 µL of resuspension solution</p>
 
-
<p> - Ribolyse three times for 60 s with 6500 rpm (30 s break between every run, 400 mg beads)</p>
 
-
<p> - Centrifuge 3 min at 10,000 rpm</p>
 
-
<p> - Transfer 500 µL of supernatant into new 2 mL tube</p>
 
-
<p> - Add 500 µL of lysis buffer, invert 4 - 6 times</p>
 
-
<p> - Add 700 µL of neutralisation buffer, invert 4 - 6 times</p>
 
-
<p> - Centrifuge 10 min at 12,000 rpm</p>
 
-
<p> - Transfer 2 x 600 µL of supernatant into spin column (centrifuge down each time for 30 s at 12,000 rpm)</p>
 
-
<p> - Two wash steps with 750 µL of washing buffer</p>
 
-
<p> - Dry column</p>
 
-
<p> - Elute in 75 µL of elution buffer</p>
 
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<br>
 
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</div>
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==Genetic Engineering==
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<div class="eccs_headline">
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===Whole Genome Isolation===
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<a name="iso_whole"><span style="color:#ff6600">[Generating electrocompetent cells]</span></a>
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*For the isolation following kit has been used: Fermentas GeneJET™ Plasmid Miniprep Kit
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<br><b><u>Material </u></b>
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<p> - 550 mL LB-Medium</p>
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-
<p> - 1 L cooled bidest. H2O</p>
+
-
<p> - 150 mL cooled 10 % glycerine</p>
+
-
<p> - 10 pre-cooled 50 mL Falcons</p>
+
 +
**Centrifuge 10 mL of over-night liquid culture
 +
**Resuspend pellet in 800 µL of resuspension solution
 +
**Ribolyse three times for 60 s with 6500 rpm (30 s break between every run, 400 mg beads)
 +
**Centrifuge 3 min at 10,000 rpm
 +
**Transfer 500 µL of supernatant into new 2 mL tube
 +
**Add 500 µL of lysis buffer, invert 4 - 6 times
 +
**Add 700 µL of neutralisation buffer, invert 4 - 6 times
 +
**Centrifuge 10 min at 12,000 rpm
 +
**Transfer 2 x 600 µL of supernatant into spin column (centrifuge down each time for 30 s at 12,000 rpm)
 +
**Two wash steps with 750 µL of washing buffer
 +
**Dry column
 +
**Elute in 75 µL of elution buffer
-
<br><b><u>Protocol: </u></b>
+
 
-
<p> - Inoculate 2x3 mL LB with bacterial stock; incubate over night at 37 °C and 200 rpm</p>
+
===Generating electrocompetent cells===
-
<p> - Inoculate 2x250 mL LB with the over night cultures in 1-litre-flask at 37 °C and 140 rpm </p>
+
*Material:
-
<p> - Incubate until OD600 0,4-0,6</p>
+
**550 mL LB-Medium
-
<p> - Cool the culture 15-30 minutes on ice</p>
+
**1 L cooled bidest. H2O
-
<p> <b>Onwards all steps at 4°C</b><p>
+
**150 mL cooled 10 % glycerine
-
<p> - Divide the cultures into cooled 50 mL Falcons and centrifugate at 4000 rpm, 4 °C for 15 minutes, make sure to slowly accelerate and deccelerate </p>
+
**10 pre-cooled 50 mL Falcons
-
<p> - Discard supernatant</p>
+
 
-
<p> - Resuspend pellet in 5 mL cooled bidest H2O (and don't get frustrated while doing it, keep shaking gently)</p>
+
 
-
<p> - Pool two suspensions each, add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)</p>
+
*Protocol:
-
<p> - Discard supernatant</p>
+
**Inoculate 2x3 mL LB with bacterial stock; incubate over night at 37 °C and 200 rpm
-
<p> - Resuspend pellet in 5 mL cooled bidest H2O</p>
+
**Inoculate 2x250 mL LB with the over night cultures in 1-litre-flask at 37 °C and 140 rpm  
-
<p> - Add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)</p>
+
**Incubate until OD600 0,4-0,6
-
<p> - Discard supernatant</p>
+
**Cool the culture 15-30 minutes on ice
-
<p> - Resuspend pellet in 5 mL cooled 10 % glycerine</p>
+
**Onwards all steps at 4°C
-
<p> - Transfer suspensions in two 50 mL Falcons and centrifugate again (see centrifugation above)</p>
+
**Divide the cultures into cooled 50 mL Falcons and centrifugate at 4000 rpm, 4 °C for 15 minutes, make sure to slowly accelerate and deccelerate
-
<p> - Discard supernatant</p>
+
**Discard supernatant
-
<p> - Add volume of 10 % glycerine that is approximately equal to the volume of the pellet and resuspend</p>
+
**Resuspend pellet in 5 mL cooled bidest H2O (and don't get frustrated while doing it, keep shaking gently)
-
<p> - Divide cells in 100 μL aliquots and freeze in liquid N2 immediately</p>
+
**Pool two suspensions each, add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)
-
<p> - Store at -80 °C</p>
+
**Discard supernatant
-
<br>
+
**Resuspend pellet in 5 mL cooled bidest H2O
 +
**Add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)
 +
**Discard supernatant
 +
**Resuspend pellet in 5 mL cooled 10 % glycerine
 +
**Transfer suspensions in two 50 mL Falcons and centrifugate again (see centrifugation above)
 +
**Discard supernatant
 +
**Add volume of 10 % glycerine that is approximately equal to the volume of the pellet and resuspend
 +
**Divide cells in 100 μL aliquots and freeze in liquid N2 immediately
 +
**Store at -80 °C
 +
 
 +
 
 +
===Transformation via electroporation===
 +
*Thaw 50 µL competent E.coli cells on ice, dilute with icecold 50 µL glycerine (10 %) if necessary
 +
*Add 0.5-5 µL plasmid to 50 µl electrocompetent cells
 +
*Store cells on ice for 1 minute
 +
*Electroporate at U = 2.5 kV, C = 25 µF, R = 400 Ώ
 +
*Transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37 °C
 +
*Centrifuge 2 min at 2000 rpm and plate on selective LB-Medium
 +
*Incubate over night at 37 °C
 +
 
 +
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<a name="e_trafo"><span style="color:#ff6600">[Transformation via electroporation]</span></a>
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<br>
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<p> - Thaw 50 µL competent E.coli cells on ice, dilute with icecold 50 µL glycerine (10 %) if necessary</p>
+
-
<p> - Add 0.5-5 µL plasmid to 50 µl electrocompetent cells</p>
+
-
<p> - Store cells on ice for 1 minute</p>
+
-
<p> - Electroporate at U = 2.5 kV, C = 25 µF, R = 400 Ώ</p>
+
-
<p> - Transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37 °C</p>
+
-
<p> - Centrifuge 2 min at 2000 rpm and plate on selective LB-Medium</p>
+
-
<p> - Incubate over night at 37 °C</p>
+
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</div>
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Revision as of 22:00, 27 September 2013



Protocols



Genetic Engineering

Whole Genome Isolation

  • For the isolation following kit has been used: Fermentas GeneJET™ Plasmid Miniprep Kit
    • Centrifuge 10 mL of over-night liquid culture
    • Resuspend pellet in 800 µL of resuspension solution
    • Ribolyse three times for 60 s with 6500 rpm (30 s break between every run, 400 mg beads)
    • Centrifuge 3 min at 10,000 rpm
    • Transfer 500 µL of supernatant into new 2 mL tube
    • Add 500 µL of lysis buffer, invert 4 - 6 times
    • Add 700 µL of neutralisation buffer, invert 4 - 6 times
    • Centrifuge 10 min at 12,000 rpm
    • Transfer 2 x 600 µL of supernatant into spin column (centrifuge down each time for 30 s at 12,000 rpm)
    • Two wash steps with 750 µL of washing buffer
    • Dry column
    • Elute in 75 µL of elution buffer


Generating electrocompetent cells

  • Material:
    • 550 mL LB-Medium
    • 1 L cooled bidest. H2O
    • 150 mL cooled 10 % glycerine
    • 10 pre-cooled 50 mL Falcons


  • Protocol:
    • Inoculate 2x3 mL LB with bacterial stock; incubate over night at 37 °C and 200 rpm
    • Inoculate 2x250 mL LB with the over night cultures in 1-litre-flask at 37 °C and 140 rpm
    • Incubate until OD600 0,4-0,6
    • Cool the culture 15-30 minutes on ice
    • Onwards all steps at 4°C
    • Divide the cultures into cooled 50 mL Falcons and centrifugate at 4000 rpm, 4 °C for 15 minutes, make sure to slowly accelerate and deccelerate
    • Discard supernatant
    • Resuspend pellet in 5 mL cooled bidest H2O (and don't get frustrated while doing it, keep shaking gently)
    • Pool two suspensions each, add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)
    • Discard supernatant
    • Resuspend pellet in 5 mL cooled bidest H2O
    • Add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)
    • Discard supernatant
    • Resuspend pellet in 5 mL cooled 10 % glycerine
    • Transfer suspensions in two 50 mL Falcons and centrifugate again (see centrifugation above)
    • Discard supernatant
    • Add volume of 10 % glycerine that is approximately equal to the volume of the pellet and resuspend
    • Divide cells in 100 μL aliquots and freeze in liquid N2 immediately
    • Store at -80 °C


Transformation via electroporation

  • Thaw 50 µL competent E.coli cells on ice, dilute with icecold 50 µL glycerine (10 %) if necessary
  • Add 0.5-5 µL plasmid to 50 µl electrocompetent cells
  • Store cells on ice for 1 minute
  • Electroporate at U = 2.5 kV, C = 25 µF, R = 400 Ώ
  • Transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37 °C
  • Centrifuge 2 min at 2000 rpm and plate on selective LB-Medium
  • Incubate over night at 37 °C





Contents

Retrieved from "http://2013.igem.org/Team:Bielefeld-Germany/Labjournal/Molecular"