Team:Colombia/Protocols

From 2014.igem.org

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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><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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===Instructions for gel preparation===
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<li>Weigh 0,3g of agarose.</li>
<li>Weigh 0,3g of agarose.</li>
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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)====
 
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<li>10 g tryptone</li>
<li>10 g tryptone</li>
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<li>Water</li>
<li>Water</li>
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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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<li>15 g agar agar</li>
<li>15 g agar agar</li>
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For selective medium, suplement with 25 ug/mL of antibiotic (kanamycin or chloramphenicol as appropiate).
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For selective medium, suplement with antibiotic as appropiate (kanamycin 50 ug/ mL and 100 uL/mL for chloramphenicol or ampicillin ).
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===Protocol for electrocompetent cells===
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<b> <font color="#8A0808" size="5" > Electrocompetent cells </font> </b>
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===Genome Extraction===
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<b> <font color="#8A0808" size="5" > Genome extraction </font> </b>
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For bacterial genome extraction we used Easy DNA Kit, Invitrogen according to <html><a href="https://2014.igem.org/File:Easy-DNA_Kit.pdf" target="_blank">manufacturer's instructions</a>.</html>
For bacterial genome extraction we used Easy DNA Kit, Invitrogen according to <html><a href="https://2014.igem.org/File:Easy-DNA_Kit.pdf" target="_blank">manufacturer's instructions</a>.</html>
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===Plasmids Extraction===
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<b> <font color="#8A0808" size="5" > Plsmids extraction </font> </b>
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For bacterial plasmid extraction we used GenElute Plasmid Miniprep Kit, Sigma Aldrich according to <html> <a href="https://2014.igem.org/File:Miniprep.pdf" target="_blank">manufacturer's instructions</a>. </html>
For bacterial plasmid extraction we used GenElute Plasmid Miniprep Kit, Sigma Aldrich according to <html> <a href="https://2014.igem.org/File:Miniprep.pdf" target="_blank">manufacturer's instructions</a>. </html>
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<b> <font color="#8A0808" size="5" > Transformation by electroporation </font> </b>
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==='''Transformation by electroporation'''===
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<li>Mix 40 uL of electrocompetent cells with 4 uL of DNA (we used iGEM BioBricks resuspended in 20 uL of miliQ water ).</li>
<li>Mix 40 uL of electrocompetent cells with 4 uL of DNA (we used iGEM BioBricks resuspended in 20 uL of miliQ water ).</li>
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<b> <font color="#8A0808" size="5" > Biobrick Assembly </font> </b>
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Revision as of 03:22, 4 October 2014

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Protocols



Agarose gel

  1. Weigh 0,3g 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 ug/ mL and 100 uL/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.



Plsmids extraction

<p align="justify"> For bacterial plasmid extraction we used GenElute Plasmid Miniprep Kit, Sigma Aldrich according to manufacturer's instructions.

Transformation by electroporation

<p align="justify">

  1. Mix 40 uL of electrocompetent cells with 4 uL of DNA (we used iGEM BioBricks resuspended in 20 uL 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 uL 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

<p align="justify">

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.

Retrieved from "http://2014.igem.org/Team:Colombia/Protocols"