Team:Colombia/Protocols

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<h2>Protocols</h2>
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<center><b><h1  class="curs1"> Protocols</h1></b></center>
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    <li class="active"><a href="#tab1" data-toggle="tab">Agarosa gel</a></li>
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    <li><a href="#tab2" data-toggle="tab">LB Medium</a></li>
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    <li><a href="#tab3" data-toggle="tab">Electrocompetent cells</a></li>
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<br><br>
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    <li><a href="#tab4" data-toggle="tab">Bacterial genome extraction</a></li>
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<b> <font color="#8A0808" size="5" > Agarose gel </font> </b>
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    <li><a href="#tab5" data-toggle="tab">Plasmids extraction</a></li>
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<br><br>
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    <li><a href="#tab6" data-toggle="tab">Transformation</a></li>
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<p align="justify">
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    <li><a href="#tab7" data-toggle="tab">Digestion and ligation</a></li>
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===Instructions for gel preparation===
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<ol>
<ol>
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<li>Weigh 0,3g of agarose.</li>
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<li>Weigh 0,3 g of agarose.</li>
<li>Add 30 mL of TAE 1X.</li>
<li>Add 30 mL of TAE 1X.</li>
<li>Heat up until the solution is homogeneous, avoiding boiling. If it boils, move away from the heat until it “calms down” and put it back on the heat until the agarose is completely dissolved.</li>
<li>Heat up until the solution is homogeneous, avoiding boiling. If it boils, move away from the heat until it “calms down” and put it back on the heat until the agarose is completely dissolved.</li>
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<li>Mix the samples with loading dye in a 5:1 ratio. Put the samples into the wells, as well as 4 µL of molecular weight marker into the first well.</li>
<li>Mix the samples with loading dye in a 5:1 ratio. Put the samples into the wells, as well as 4 µL of molecular weight marker into the first well.</li>
</ol>
</ol>
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Note: For big gels use 0,7 g of agarose, 70 mL of TAE 1x and 3 µL of SYBR SAFE Gel Stain.
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<b>Note</b>: For big gels use 0,7 g of agarose, 70 mL of TAE 1x and 3 µL of SYBR SAFE Gel Stain.
   
   
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<b> <font color="#8A0808" size="5" > LB medium (1L liquid) </font> </b>
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===Instructions for LB medium preparation===
 
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====LB liquid medium preparation (1 L)====
 
<ul>
<ul>
<li>10 g tryptone</li>
<li>10 g tryptone</li>
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<li>Water</li>
<li>Water</li>
</ul>
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====LB solid medium preparation (1 L)====
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<b> <font color="#8A0808" size="5" > LB medium (solid, 1L = 50 dishes) </font> </b>
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<ul>
<ul>
<li>15 g agar agar</li>
<li>15 g agar agar</li>
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<li>Water</li>
<li>Water</li>
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For selective medium, suplement with antibiotic as appropiate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin ).
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For selective medium, suplement with 25 ug/mL of antibiotic (kanamycin or chloramphenicol as appropiate).
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<b> <font color="#8A0808" size="5" > Electrocompetent cells </font> </b>
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===Protocol for electrocompetent cells===
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<ol>
<ol>
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<li>Divide the ON culture in 50 mL falcon tubes.</li>
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<li>Divide the ON culture in 50 mL falcon tubes (we always used ''E. coli'' TOP10).</li>
<li>Centrifuge 8000 rpm x 10 min.</li>
<li>Centrifuge 8000 rpm x 10 min.</li>
<li>Discard supernatant.</li>
<li>Discard supernatant.</li>
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<li>Centrifuge again.</li>
<li>Centrifuge again.</li>
<li>Discard supernatant.</li>
<li>Discard supernatant.</li>
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<li>Resuspend with glycerol with water. Glycerol 10%.</li>
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<li>Resuspend with glycerol with water. Glycerol 10 %.</li>
<li>Centrifugue.</li>
<li>Centrifugue.</li>
<li>Repeat steps 8-10.</li>
<li>Repeat steps 8-10.</li>
<li>Discard supernatant and divide what is left in eppendorfs.</li>
<li>Discard supernatant and divide what is left in eppendorfs.</li>
</ol>
</ol>
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CAUTION: Everything must be done on ice (0-3°C) (reactants, centrifuge, transport, containers).
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<b>CAUTION</b>: Everything must be done on ice (0-3°C) (reactants, centrifuge, transport, containers).
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<br><br><br>
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<b> <font color="#8A0808" size="5" > Genome extraction </font> </b>
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<br><br>
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<p align="justify">
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For bacterial genome extraction we used Easy DNA Kit, Invitrogen according to <a href="https://2014.igem.org/File:Easy-DNA_Kit.pdf" target="_blank">manufacturer's instructions</a>.
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<b> <font color="#8A0808" size="5" > Plasmids extraction </font> </b>
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<p align="justify">
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For bacterial plasmid extraction we used GenElute Plasmid Miniprep Kit, Sigma Aldrich according to  <a href="https://2014.igem.org/File:Miniprep.pdf" target="_blank">manufacturer's instructions</a>.
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===Genome Extractio===
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<br><br><br>
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For bacterial genome extraction we used Easy DNA Kit, Invitrogen according to manufacturer's instructions:[[File:Easy-DNA Kit.pdf|Easy-DNA Kit.pdf]]
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<b> <font color="#8A0808" size="5" > Transformation by electroporation </font> </b>
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===Plasmids Extraction===
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For bacterial plasmid extraction we used PureYield Plasmid Miniprep System, Promega according to manufacturer's instructions:[[File:Miniprep.pdf|Miniprep.pdf]]
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==='''Transformation by electroporation'''===
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<ol>
<ol>
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<li>Mix 40 uL of electrocompetent cells with 4 uL of DNA (we used resuspended iGEM biobricks)</li>
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<li>Mix 40 µL of electrocompetent cells with 4 µL of DNA (we used iGEM BioBricks resuspended in 20 µL of miliQ water ).</li>
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<li>Incube the cuvettes for electroporation on ice and the above mix as well</li>
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<li>Incube the cuvettes for electroporation on ice and the above mix as well.</li>
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<li>Electroporate on the cuvette</li>
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<li>Electroporate into the cuvette.</li>
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<li>Add (as quick as possible) 200 uL of LB medium</li>
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<li>Add (as quick as possible) 200 µL of LB medium.</li>
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<li>Incubate for 30 min at 37 °C </li>
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<li>Incubate for 30 min at 37 °C .</li>
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<li>Plate bacteria over sumplemented LB medium</li>
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<li>Plate bacteria over sumplemented LB medium.</li>
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<li>Incubate at 37 °C, 24 h</li>
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<li>Incubate at 37 °C, 24 h.</li>
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<li>Use isolated colonies to check the correct insertion</li>
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<li>Use isolated colonies to check the correct insertion.</li>
</ol>
</ol>
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<b> <font color="#8A0808" size="5" > Biobrick Assembly </font> </b>
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==='''COTENIDO  7'''===
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Lalalala
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The digestion and ligation of the BioBricks were carried out using the Biobrick Assembly Kit, New England Biolabs Inc. The protocols for both procedures can be found <a href="http://www.neb.com/products/e0546-biobrick-assembly-kit" target="_blank">here</a>.
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<div class="button-fill orange" ><div class="button-text">Back to Wet Lab</div><div class="button-inside"><div class="inside-text"><a style="text-decoration: none; background-color: none; color: red;" href="https://2014.igem.org/Team:Colombia/WetLab">Go! </a></div></div></div>
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Latest revision as of 04:32, 11 October 2014

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Protocols



Agarose gel

  1. Weigh 0,3 g of agarose.
  2. Add 30 mL of TAE 1X.
  3. Heat up until the solution is homogeneous, avoiding boiling. If it boils, move away from the heat until it “calms down” and put it back on the heat until the agarose is completely dissolved.
  4. While heating, prepare the bed in which the gel will polymerize. Make sure that it is well balanced and tight, and that the “comb” is well placed.
  5. When homogeneous, add 2 µL of SYBR SAFE DNA Gel Stain to the solution and mix well.
  6. Pour the solution into the bed and clear all its bubbles with a tip. Put a piece of paper on top of it and let it polymerize.
  7. Mix the samples with loading dye in a 5:1 ratio. Put the samples into the wells, as well as 4 µL of molecular weight marker into the first well.
Note: For big gels use 0,7 g of agarose, 70 mL of TAE 1x and 3 µL of SYBR SAFE Gel Stain.


LB medium (1L liquid)

  • 10 g tryptone
  • 10 g NaCl
  • 5 g yeast extract
  • Water



LB medium (solid, 1L = 50 dishes)

  • 15 g agar agar
  • 10 g tryptone
  • 10 g NaCl
  • 5 g yeast extract
  • Water

For selective medium, suplement with antibiotic as appropiate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin ).


Electrocompetent cells

  1. Divide the ON culture in 50 mL falcon tubes (we always used ''E. coli'' TOP10).
  2. Centrifuge 8000 rpm x 10 min.
  3. Discard supernatant.
  4. Resuspend everything with water in two falcons.
  5. Centrifuge again.
  6. Discard supernatant and resuspend with water. Wash with water two more times.
  7. Centrifuge again.
  8. Discard supernatant.
  9. Resuspend with glycerol with water. Glycerol 10 %.
  10. Centrifugue.
  11. Repeat steps 8-10.
  12. Discard supernatant and divide what is left in eppendorfs.
CAUTION: Everything must be done on ice (0-3°C) (reactants, centrifuge, transport, containers).


Genome extraction

For bacterial genome extraction we used Easy DNA Kit, Invitrogen according to manufacturer's instructions.


Plasmids extraction

For bacterial plasmid extraction we used GenElute Plasmid Miniprep Kit, Sigma Aldrich according to manufacturer's instructions.


Transformation by electroporation

  1. Mix 40 µL of electrocompetent cells with 4 µL of DNA (we used iGEM BioBricks resuspended in 20 µL of miliQ water ).
  2. Incube the cuvettes for electroporation on ice and the above mix as well.
  3. Electroporate into the cuvette.
  4. Add (as quick as possible) 200 µL of LB medium.
  5. Incubate for 30 min at 37 °C .
  6. Plate bacteria over sumplemented LB medium.
  7. Incubate at 37 °C, 24 h.
  8. Use isolated colonies to check the correct insertion.



Biobrick Assembly

The digestion and ligation of the BioBricks were carried out using the Biobrick Assembly Kit, New England Biolabs Inc. The protocols for both procedures can be found here.

Back to Wet Lab