Team:Colombia/Protocols

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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 in this link: [https://www.neb.com/products/e0546-biobrick-assembly-kit#tabselect2]
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Revision as of 00:36, 26 June 2014

Protocols

Contents

Instructions for gel preparation

  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.


Instructions for LB medium preparation

LB liquid medium preparation (1 L)

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

LB solid medium preparation (1 L)

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

For selective medium, suplement with 25 ug/mL of antibiotic (kanamycin or chloramphenicol as appropiate).


Protocol for electrocompetent cells

  1. Divide the ON culture in 50 mL falcon tubes.
  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:File:Easy-DNA Kit.pdf


Plasmids Extraction

For bacterial plasmid extraction we used PureYield Plasmid Miniprep System, Promega according to manufacturer's instructions:File:Miniprep.pdf



Transformation by electroporation

  1. Mix 40 uL of electrocompetent cells with 4 uL of DNA (we used resuspended iGEM biobricks)
  2. Incube the cuvettes for electroporation on ice and the above mix as well
  3. Electroporate on 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


Biobricks 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 in this link: [https://www.neb.com/products/e0546-biobrick-assembly-kit#tabselect2]



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