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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

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
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
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

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
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
Transformation
1. Transform up to 20uL of the ligation mix according to standard E.coli transformation protocol

Preparation of chemically competent cells CCMB 80 method

This protocol has been used with the TOP10 and Mach1 E. coli cell strains from Invitrogen

Preparation

- All glassware MUST be detergent free, detergents severely inhibit transformation efficiency of competent cells. An easy way to ensure that glassware is detergent free is to autoclave it half full with ddH¬2O.
- All glassware and tubes used for the actual generation of the competent cells MUST be pre-chilled

Generation of seed stocks

Streak TOP10 cells on an SOB plate and grow for single colonies at room temperature.
Pick single colonies into 2 ml of SOB medium and shake overnight at room temperature.
Add glycerol to 15% (vol) and aliquot 250 μL samples to -80 °C freezer tubes.
Store at -80 °C indefinitely.

Preparing competent cells

Inoculate 250 μL seed stock into a 500 mL Erlenmeyer flask containing 50 mL SOB medium. Grow at room temperature until a cell density of 5 × 107 cells/ml is reached.
Collect the cell suspension into a sterile 50 mL centrifuge tube and chill on ice for 10 min. (Take a 10 μL aliquot to determine the density of viable cells by plating a 106 dilution onto a SOB agar plate).
Pellet the cells at 1000 g for 10 min at 4°C. Decant the supernatant and invert the tubes to remove excess culture medium. Resuspend the cells in 17 mL of CCMB 80 by gentle vortexing of the centrifuge tube.
Incubate on ice for 20 min. Centrifuge for 10 min at 1000 g at 4°. Decant as in Step 3. Resuspend the cells in 4 mL of CCMB 80.
Aliquot 200 μL volumes of the cells into prechilled eppendorf tubes and then stored at -80 °C.

Materials

CCMB 80 (500 mL)

Compound Amount/500 mL Final concentration CaCl2*2H2O 2.95 g 80 mM MnCl2*4H2O 1.0 g 20 mM MgCl2*6H2O 0.5 g 10 mM potassium acetate 2.5 mL of a 1 M stock (pH 7.0) 10 mM glycerol 25 mL 10% (v/v)

Preparation: First prepare a 1 M solution of potassium acetate and adjust to pH 7.0 using KOH, then sterilize by filtration through a 0.2 μm membrane and store frozen. Then prepare a solution of 10 mM potassium acetate, 10% glycerol (v/v) using these reagents and the purest available water. Add salts as solids and allow each to enter into solution before adding the next. Adjust pH to 6.4 with 0.1 N HCl. Do not adjust pH upward with base. Sterilize the solution by filtration through a 0.22 μm filter and store at 4°.

SOB Media (500 mL)

Compound Amount / 500 mL Final concentration yeast extract 2.5 g 0.5% (w/v) tryptone 10 g 2% (w/v) NaCl 0.292 g 10 mM KCl 0.093 g 2.5 mM MgSO4 7H2O 2.45 g 20 mM

Preparation: Combine all ingredients and add water to a final volume of 500 mL. Adjust to pH 7.5, this requires approximately 25 ml of 1M NaOH per liter. Autoclave for 45 minutes.

References:

[1] http://openwetware.org/wiki/TOP10_chemically_competent_cells [2] Hanahan et al. [4] Plasmid transformation of Escherichia coli and other bacteria. Methods in Enzymology (1991) vol. 204 pp. 63-114

Yeast transformation

Followed the exact protocol found in: Gietz and Schiestl. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc (2007) vol. 2 (1) pp. 31-34

Team:uOttawa/Notebook