Team:Bielefeld-Germany/Labjournal/Molecular

From 2013.igem.org

(Difference between revisions)
m
m
Line 14: Line 14:
     $(".iso_whole").toggle();
     $(".iso_whole").toggle();
   });
   });
-
   $(".cyt_frag1_headline").click(function(){
+
   $(".eccs_headline").click(function(){
-
     $(".cyt_frag1").toggle();
+
     $(".eccs").toggle();
   });
   });
   $(".cyt_frag2_headline").click(function(){
   $(".cyt_frag2_headline").click(function(){
Line 30: Line 30:
<style type="text/css">
<style type="text/css">
.iso_whole{display:none;}
.iso_whole{display:none;}
-
.cyt_frag1{display:none;}
+
.eccs1{display:none;}
.cyt_frag2{display:none;}
.cyt_frag2{display:none;}
-
.h1{color:#ff6600;}
 
</style>
</style>
Line 42: Line 41:
<br>
<br>
<!--<h2>Standard Protocols</h2> -->
<!--<h2>Standard Protocols</h2> -->
 +
<div class="iso_whole_headline">
<div class="iso_whole_headline">
<a name="iso_whole"><span style="color:#ff6600">[Whole Genome Isolation]</span></a>
<a name="iso_whole"><span style="color:#ff6600">[Whole Genome Isolation]</span></a>
-
 
-
 
-
 
-
 
-
 
</div>
</div>
<div class="iso_whole">
<div class="iso_whole">
Line 69: Line 64:
<br>
<br>
 +
</div>
-
<!--
+
<div class="eccs_headline">
-
<p> -</p>
+
<a name="iso_whole"><span style="color:#ff6600">[Generating electrocompetent cells]</span></a>
-
<p> -</p>
+
-
<p> -</p>
+
-
<p> -</p>
+
-
-->
+
-
<br>
+
</div>
</div>
 +
<div class="eccs">
 +
<br><b><u>Material </u></b>
 +
<p> - 550 mL LB-Medium</p>
 +
<p> - 1 L cooled bidest. H2O</p>
 +
<p> - 150 mL cooled 10 % glycerine</p>
 +
<p> - 10 pre-cooled 50 mL Falcons</p>
-
<br><br><br><br><br><br><br><br><br><br><br><br><br>
 
 +
<br><b><u>Protocol: </u></b>
 +
<p> - Inoculate 2x3 mL LB with bacterial stock; incubate over night at 37 °C and 200 rpm</p>
 +
<p> - Inoculate 2x250 mL LB with the over night cultures in 1-litre-flask at 37 °C and 140 rpm </p>
 +
<p> - Incubate until OD600 0,4-0,6</p>
 +
<p> - Cool the culture 15-30 minutes on ice</p>
 +
<p> <b>Onwards all steps at 4°C</b><p>
 +
<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>
 +
<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>
 +
<p> - Discard supernatant</p>
 +
<p> - Resuspend pellet in 5 mL cooled bidest H2O</p>
 +
<p> - Add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)</p>
 +
<p> - Discard supernatant</p>
 +
<p> - Resuspend pellet in 5 mL cooled 10 % glycerine</p>
 +
<p> - Transfer suspensions in two 50 mL Falcons and centrifugate again (see centrifugation above)</p>
 +
<p> - Discard supernatant</p>
 +
<p> - Add volume of 10 % glycerine that is approximately equal to the volume of the pellet and resuspend</p>
 +
<p> - Divide cells in 100 μL aliquots and freeze in liquid N2 immediately</p>
 +
<p> - Store at -80 °C</p>
 +
 +
<br>
</div>
</div>
 +
</body>
</body>
</html>
</html>
 +
 +
<!--
 +
<p> -</p>
 +
<p> -</p>
 +
<p> -</p>
 +
<p> -</p>
 +
-->

Revision as of 21:48, 25 September 2013






Genetic enginering protocols


[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



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