Protocol

Contents

• MATERIALS
  • • LB medium
  • • LB agar
  • • Agarose gel
• METHODS
  • • Preparation of chemically competent E. coli
  • • Transformation of chemically competent E. coli
  • • Preparation of electro competent S. cerevisiae
  • • Transformation of electro competent S. cerevisiae
  • • Plasmid DNA Extraction
  • • PCR Clean-Up/ Gel Extraction
  • • Q5 PCR
  • • OneTaq colony PCR
  • • Golden Gate
  • • mRNA extraction
  • • cDNA synthesis

Materials

LB medium

LB medium is a standard liquid medium for the cultivation of E. coli.

The LB medium is sterilized via autoclavation. After cooling down antibiotics may be added.

LB agar

LB agar is a standard agar for the cultivation of E. coli.

The LB agar is sterilized via autoclavation. After cooling down to approximately 60 °C antibiotics may be added. The LB agar is poured into disposable petri dishes.

Agarose gel

Agarose gels are used for a gel electrophoresis.

• Heat 500 mL flask in microwave until liquid is completely clear.
• Add 8µL of Midori Green.
• Pour liquid into 3 prepared gel casts with 2 combs.
• Gel solidifies after approximately 30 mins.

Methods

Preparation of chemical competent E. coli

• E. coli strain DH10B is inoculated from a glycerol stock onto an LB plate; the inoculum is streaked on the plate using a loop so as to obtain individual colonies. The plate is incubated overnight at 37 °C.
• Inoculate 10 mL of LB medium with a single colony and incubate the flask overnight in a shaker-incubator (37°C, shaking 180 rpm).
• The following day, transfer 2 mL of this culture to a flask containing 200 mL LB medium and incubate for about 2h until OD₆₀₀ raches 0.6.
• Cool down the cells on ice for 10 minutes. The cells are pelleted in a centrifuge for 5 min at 4,500 rpm (4,000 x g) at 4°C. The cells are resuspended in 0.4 volume of ice-cold TFB1*.
• Repeat the centrifugationstep. Resuspend the pellet in 1/25 volume of ice-cold TFB2**.
• the cells are aliquoted 100 µL per tube and stored at -80 °C.

* Solution TFB1: 30 mM potassium acetate, 10 mM CaCl₂, 50 mM MnCl₂, 100 mM RbCl, and 15% glycerol; adjust to pH 5.8 (with 1 M acetic acid), filter-sterilize, and store at 4 °C (ready to use) or at room temperature (cool down before use).
** Solution TFB2: 100 mM MOPS (or PIPES), 75 mM CaCl₂, 10 mM RbCl, and 15% glycerol; adjust to pH 6.5 (with 1 M KOH), filter-sterilize, and store at 4 °C (ready to use) or at room temperature (cool down before use).

Transformation of chemical competent E. coli

• Frozen chemically competent cells (100 µL per tube) are thawed on ice.
• The entire ligation (or other DNA sample to be transformed) is added to the cells, and the mix incubated on ice for 30 minutes.
• The cells and DNA mix is heat shocked for 90 s at 42 °C.
•Allow the cells to recover on ice for 5 minutes.
• Add 1 mL of LB medium to the cells and incubate the tube at 37 °C in a shaker-incubator (150 rpm) for 45 min to 1h.
• Plate the appropriate amount of cells on selective LB agar.

Preparation of electro competent S. cerevisiae

Please make sure that the sorbitol (1M) for usage after pre-treatment is ice-cold before you start. Also, take care that your cells are kept ice-cold throughout the whole procedure, from pre-treatment until freezing the final aliquots at - 80 °C.
Day 1 (evening)
• Prepare the pre-culture: inoculate 5 - 10 mL selective YPD medium with a fresh colony (no older than 3 weeks) and incubate overnight (o/n; ~16 h) (shaker; 180 rpm; 25 °C).
Day 2 (evening)
• Main culture: Inoculate 100 mL non-selective YPD medium with the preculture and incubate o/n (~16 h) (shaker; 180 rpm; 25 °C); the end-OD should be between 1.2 and 2.5. Calculate the volume of pre-culture needed for inoculation of the main culture.
Day 3
• Measure OD and split 90 mL of the culture into 2 Falcon-tubes (2 x 45 mL).
• Harvest the cells by centrifuging (5 min; 1500 xg and 4 °C), then discard the supernatant.
• Add 10 mL of pre-treating solution* (25 °C) to each of the pellets and resuspend the cells.
• Incubate the cells for 30 min (Shaker; 180 rpm; 25 °C).
• Add 40 mL ice-cold sorbitol (1 M) to each Falcon-tube and harvest cells (5 min; 1500 xg; 4 °C), then discard supernatant.
• Combine pellets in one Falcon-tube and resuspend cells in 45 mL ice-cold sorbitol (1 M).
• Harvest the cells (5 min; 1500 xg; 4 °C), then discard supernatant.
• Resuspend in cells in 200 µL ice-cold sorbitol (1 M); aliquot cells into pre-chilled Eppendorf tubes (á 100 µL) and keep them on ice until transformation (long term storage: - 80 °C).

* pre-treating solution (20 mL are needed for one 100mL cell batch): 7.4 mL AD; 12 mL of 1 M Sorbitol (for a final concentration of 0.6 M); 200 µL of 1 M Tris-HCl, pH 7.5 (for a final concentration of 10 mM); 200 µL of 1 M DTT (for a final concentration of 10 mM); 200 µL of 10 M Lithium acetate (for a final concentration of 100 mM)

Transformation of electro competent S. cerevisiae

• An 100 µL aliquot of the electro-competent S. cerevisiae cells is mixed very gently with a small volume (use up to 30 µL eluate after column purification, see PCR Clean-Up/ Gel Extraction) of the linearized DNA. As a negative control, cells should be transformed with the elution solution or AD. The mixture should not be vortexed to avoid shearing of the DNA and cells.
• The mixture is then transferred into a chilled electroporation cuvette (2 mm) and incubated on ice for 5 minutes.
• Electroporation is performed at the BioRad MicroPulser^TM using following parameters: 2000 V, 25 µF and 186 Ω.
• Immediately after transformation 1 mL of ice-cold YPD-media (or 1 M sorbitol for His-selection) is added to the electroporated cells in the cuvette, then the content of the cuvette is transferred carefully into a sterile microcentrifuge tube.
• Regnerate the cells for at least 1.5 h to maximum3h at 28 °C before plating aliquots (eg 20 µL, 200 µL) on selective agar plates. Keep the rest at 4 °C.
• Incubate the plates at 28 °C until colonies appear (approx. 48 h).

Plasmid DNA extraction

Plasmid DNA extractions are performed using Hi Yield^® Plasmid MINI kit according to the manufacturer's protocol .

PCR Clean-up/ Gel Extraction

PCR clean-ups and Gel extractions are performed using Hi Yield^® PCR Clean-up/ Gel Extractions Kit according to the manufacturer's protocol .

Q5 PCR

Vortex and spin down PCR tube. Put tube in a thermocycler.

Thermocycler settings:

Add 5 µL of 6x Loading Dye for gel electrophoresis.

OneTaq colony PCR

Vortex and spin down PCR tube. Put tube in a thermocycler.

Thermocycler settings:

PCR Mix is ready for electrophoresis. No loading dye is needed.

Golden Gate Assembly with BsaI/ BpiI

For 20µL reaction volume pipette in a PCR Tube:

Vortex and spin down PCR tube. Put tube in a thermocycler.

Golden Gate Mix is ready for transformation.

mRNA extraction

Sample preparation:
• Centrifuge 10 mL of E. coli overnight culture and discard the supernatant.
• Add 1 mL of TRI REAGENT (Ambion, AM9738, stored dark at 4°C) to the E. coli pellet, then add approximately 500 µL glass-beads in a sealed tube.
• Ribolyze once at speed 5.5. m/s for 40 seconds.
• Allow samples to stand for 5 minutes at room temperature.
• Add 200 µL of chloroform per 1 mL of TRI REAGENT and shake vigorously for 15 seconds.
• Allow the samples to stand for 10 - 15 minutes at room temperature. Centrifuge at full speed for 10 - 15 minutes at 4 °C to separate phases [centrifugation separates the mixture into 3 phases: a red organic phase (containing protein), an interphase (containing DNA), and a colorless upper aqueous phase (containing RNA)].

RNA isolation
RNA precipitation:
• Transfer the aqueous phase to a fresh tube and add 0.5 mL of isopropanol per 1 mL of TRI REAGENT used for the initial homogenization. Take care not to transfer the interphase or the red DNA phase.
• Allow the sample to stand for 5 - 10 minutes at room temperature.
• Centrifuge at full speed for 10 minutes at 4'C. The RNA precipitate will form a pellet on the side and bottom of the tube.
RNA wash:
• Remove the supernatant and wash the RNA pellet by adding 1 mL (minimum) of 75 % ethanol per 1 mL of TRI REAGENT used for the initial homogenization.
• Vortex the sample.
• Centrifuge at full speed for 5 minutes at 4 °C.
RNA solubilization:
• Briefly dry the RNA pellet by air-drying. [Do not let the RNA pellet dry completely, as this will greatly decrease its solubility. Do not dry the RNA pellet by centrifugation under vacuum].
• Add an appropriate volume (50 - 100 µL) of nuclease-free water.
• To facilitate dissolution, mix by repeated pipetting with a micropipette and incubationg at 65 °C for 10 - 15 minutes.

cDNA synthesis

cDNA synthesis is performed using Biozym cDNA Synthesis kit according to the manufacturer's protocol with gene-specific primers.