Team:Wageningen UR/Project/ProtocolsProj1

Protocols

Media

LB

Ingredients:

10 g Bacto-tryptone

5 g yeast extract

10 g NaCl

SOB

Ingredients

0.5% (w/v) yeast extract

2% (w/v) tryptone

10 mM NaCl

2.5 mM KCl

20 mM MgSO4

Per liter

5 g yeast extract

20 g tryptone

0.584 g NaCl

0.186 g KCl

2.4 g MgSO4

SOC

Ingredients

SOB

20 mM glucose

CCMB80 buffer for preparation of chemically competent cells

Materials

Detergent-free, sterile glassware and plasticware

Table-top OD600nm spectrophotometer

SOB

CCMB80 buffer

10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)

80 mM CaCl2.2H2O (11.8 g/L)

20 mM MnCl2.4H2O (4.0 g/L)

10 mM MgCl2.6H2O (2.0 g/L)

10% glycerol (100 ml/L)

Adjust pH DOWN to 6.4 with 0.1N HCl if necessary

Adjusting pH up will precipitate manganese dioxide from Mn containing solutions

Filter sterilize and store at 4°C

Slight dark precipitate appears not to affect its function

Cloning Procedures

Preparation of chemically competent cells

Preparing glassware and media

Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic must be detergent free for these protocols. The easiest way to do this is to avoid washing glassware, and simply rinse it out. Autoclaving glassware filled 3/4 with DI water is an effective way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware.

Prechill plasticware and glassware

Prechill 250mL centrifuge tubes and screw cap tubes before use.

Preparing seed stocks

Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C works well. Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C. Add glycerol to 15% and aliquot 1 ml samples into cryotubes. Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes. Place in -80°C freezer indefinitely.Preparing competent cells. Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3. This takes approximately 16 hours.

Preparing competent cells

Aim for lower, not higher OD if you can't hit this mark.Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle.Gently resuspend in 80 ml of ice cold CCMB80 buffer. Incubate on ice 20 minutes, centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer.Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells. Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test. Incubate on ice for 20 minutes. Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates. Store at -80°C indefinitely

Test competence (see below)

Do not thaw and refreeze, as this dramatically decreases the transformation efficiency.

Transform 50 μl of cells with 1 μl of standard pUC19 plasmid. Use a 10 pg/μl stock. Hold on ice 0.5 hours. Heat shock 60 sec at 42C and add 250 μl SOC.

Incubate at 37 C for 1 hour in 2 ml centrifuge tubes under rotation.

To increase yields, an incubation time of 2 hours can be used. Plate 20 μl on AMP plates. Good cells should yield around 100 - 400 colonies. Transformation efficiency is (dilution factor=15) x colony count x 105/µg DNA. The transformation efficiency should be between 5x10^8 and 5x10^9 cfu/µg DNA.

Transformation of chemically competent cells

1. Thaw 25 - 200 μL chemically competent cells on ice. Do not use glass tubes, which adsorb DNA.

2. Add DNA (1-2 μL), pipette gently to mix

3. Incubate on ice for 30 minutes

4. Incubate cells for 30 seconds at 42°C.

5. Incubate cells on ice for 2 min.

6. Add 4 volumes of room temperature SOC (not critical)

7. Incubate for 1 hour at 37°C on shaker.

8. Spread 100-300 μl onto a plate made with appropriate antibiotic.

9. Grow overnight at 37°C.

Plasmid digestion

For digestions, Fermentas restriction kits were used according to their protocols.

Ligations

For ligations, the Fermentas T4 Ligase kit was used according to its protocol.

Gel electrophoresis

In general, 1% agarose gels were ran for 1h at 100V in 1x TAE buffer. 20 µL of sample was mixed with 6x Fermentas loading dye. 3 µL Fermentas 1kb marker was also loaded.

Gel extraction

For the extraction of fragments from agarose gels, the Fermentas Gel Extraction kit was used according to the provided protocol.

Fluorescence measurements

The part verifications were done with a microplate reader: a Molecular Devices Spectramax M2 [1] that is capable of measuring fluorescence. For these measurements 96-wells opaque Corning plates were used. 10 mL overnight cultures were grown in LB + antibiotics and spun down. As LB yielded to much background signal, the cells were resuspended in PBS to an OD600 of 0.8. 150 µL of the cell suspension was added to the wells in the plate, leaving 50 µL for additives (AHL for example). The final OD600 was 0.6 (200 µL).

For GFP measurements, the excitation wavelength of the microplate reader was set at 485 nm and the emission wavelenght at 510 nm. Measurements were done every 5 min., after 10 seconds of shaking. The temperature was maintained at 25°C to facilitate GFP maturation. The experiment was stopped if sufficient data was generated. The experiments were controlled by the program SoftMax Pro.

The AHL stock solutions were prepared by dissolving 3-oxohexanoyl-homoserine lactone in pure DMSO (due to low solubility in water). The desired concentrations were obtained by diluting these stocks in 1x PBS.

Links and references:

[http://www.moleculardevices.com/documents/general-documents/mkt-collateral/microplate-mm-collateral/SpectraMax%20M2-M2e%20datasheet%20rev%20G.pdf Molecular Devices Spectramax M2]

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