Protocols

Contents

• MATERIALS
  • LB medium
  • LB agar
• METHODS
  • Preparation of chemically competent E. coli
  • Transformation of chemically competent E. coli
  • Preparation of electro competent S. cerevisiae
  • Plasmid DNA Extraction
  • Q5 PCR
  • oneTaq colony PCR
  • Golden Gate
  • OneTaq Colony PCR

Materials

LB medium

Name	Amount
Peptone	10 g
Yeast Extract	5 g
NaCl	5 g
RO-water	1000 mL

LB agar

Name	Amount
Peptone	10 g
Yeast Extract	5 g
NaCl	5 g
RO water	986 mL
Agar	14 g

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 .

Q5 PCR

Name	Amount
oneTaq 2x MM	12.5 µL
AD	variable
Primer forward	1.25 µL
Primer reverse	1.25 µL
Template	1 ng
Total Volume	25 µL

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

PCR programme: NebQ5

Step	Temperature in °C	Time
1	98	30''
2	98	10''
3	variable - Primer annealing temperature	30''
4 → 2 30x	72	variable - 30'' /kb
5	72	3'
6	23	10''

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