Protocols

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

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.

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.