Team:uOttawa/Notebook

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Contents

Protocols

Dip and swirl cloning

This protocol is essentially the same as the Standard BioBrick Cloning protocol used in our lab with the exception of using a minimal amount of restriction enzymes.

  • Restriction Digest
    1. Add 0.3 μl of 10X NEBbuffer 2
    2. Add 0.3 μl 0f 10X BSA
    3. Add 30 ng of DNA (plasmid or PCR product)
    4. Dip the pipette tip in the enzyme solution and move it around (swirl). Place the tip in your reaction mixture, move it around (swirl) again, and rinse it.
    5. Add water to a final volume of 3 μl.
  • The remaining steps of the protocol are identical to that of the Standard BioBrick Cloning protocol.

N.B. This protocol is particularly suitable for when the enzyme solution has almost run out, but you still need to perform a number of digestions. This protocol is longer than the Standard BioBrick Cloning protocol. In addition, if the level of the enzyme solution is high, the capillarity action causes more than enough of the enzyme to enter the tip, hence, rendering this approach pointless.

Yeast Colony PCR

This is a quick extraction of yeast genomic DNA for performing PCR amplification.

  • Transfer one yeast colony, in sterile conditions, to a 50 μl solution containing the following: 3 μl of 10 mg/ml zymolase and 47 μl water.
  • Suspend the colony in the solution using a pipette.
  • Incubate the cell suspension for 30 minutes at 37°C.
  • Denature the enzyme by incubating the suspension for 10 minutes at 95°C.
  • 5 μl of the cell suspension should be used in a 50 μl PCR reaction mixture.

10 mg/ml zymolase solution

  • Add the following to a 15 mL tube,
    • 100 mg of Zymolase 20T
    • 0.5 ml of sterile Tris-HCl with pH 7.5
    • 4.5 ml of ddH2O
  • Shake gently to dissolve.
  • Add 5 ml of 50% (w/v) filter-sterilized glycerol.
  • Make aliquots depending on your need and store at -20°C.


Standard uOttawa BioBrick cloning protocol

Method A: 1 BioBrick and 1 standard BioBrick backbone

  1. Digestion
    1. Determine the DNA concentration of your desired BioBrick part and a standard BioBrick backbone (pSB1A3, pSB1C3, or pSB1T3)
    2. Combine:
      • 100ng of your desired BioBrick DNA
      • 1uL of BSA
      • 1uL of NEB Buffer 2
      • 1uL of EcoRI
      • 1uL of PstI
      • Additional ddH2O to make a final volume of 10uL
    3. Combine:
      • 100ng of a standard BioBrick backbone (pSB1A3, pSB1C3, or pSB1T3)
      • 1uL of BSA
      • 1uL of NEB Buffer 2
      • 1uL of EcoRI
      • 1uL of PstI
      • Additional ddH2O to make a final volume of 10uL
    4. Incubate the reaction mixture at 37°C for 10 minutes
    5. Heat inactivate the reaction mixture by incubating at 80°C for 20 minutes
  2. Ligation
    1. Combine:
      • 3uL of the digested BioBrick mixture
      • 3uL of the digested BioBrick backbone mixture
      • 11.5uL of ddH2O
      • 1uL of T4 ligase buffer
      • 0.5uL of T4 ligase for a total reaction volume of 20uL
    2. Incubate the reaction mixture at room temperature (25°C) for 20 minutes
    3. Heat inactivate the reaction mixture by incubating at 80°C for 20 minutes
  3. Transformation
    1. Transform up to 20uL of the ligation mix according to standard E.coli transformation protocol

Method B: 2 BioBricks (upstream and downstream parts) and 1 standard BioBrick backbone

  1. Digestion
    1. Determine the DNA concentration of your desired upstream and downstream BioBrick parts and a standard BioBrick backbone (pSB1A3, pSB1C3, or pSB1T3)
    2. Combine:
      • 100ng of your upstream BioBrick DNA
      • 1uL of BSA
      • 1uL of NEB Buffer 2
      • 1uL of EcoRI
      • 1uL of SpeI
  • Additional ddH2O to make a final volume of 10uL
    1. Combine:
      • 100ng of your downstream BioBrick DNA
      • 1uL of BSA
      • 1uL of NEB Buffer 2
      • 1uL of XbaI
      • 1uL of PstI
  • Additional ddH2O to make a final volume of 10uL
    1. Combine:
      • 100ng of a standard BioBrick backbone (pSB1A3, pSB1C3, or pSB1T3)
      • 1uL of BSA
      • 1uL of NEB Buffer 2
      • 1uL of EcoRI
      • 1uL of PstI
      • Additional ddH2O to make a final volume of 10uL
    2. Incubate the reaction mixture at 37°C for 10 minutes
    3. Heat inactivate the reaction mixture by incubating at 80°C for 20 minutes
  2. Ligation
    1. Combine:
      • 2uL of the digested upstream BioBrick mixture
      • 2uL of the digested downstream BioBrick mixture
      • 2uL of the digested backbone mixture
      • 11uL of ddH2O
      • 1uL of T4 ligase buffer
      • 1uL of T4 ligase for a total reaction volume of 20uL
    2. Incubate the reaction mixture at room temperature (25°C) for 20 minutes
    3. Heat inactivate the reaction mixture by incubating at 80°C for 20 minutes
  3. Transformation
    1. Transform up to 20uL of the ligation mix according to standard E.coli transformation protocol
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