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
  • Transformation of electro competent S. cerevisiae
  • Plasmid DNA Extraction
  • PCR Clean Up
  • Q5 PCR
  • OneTaq colony PCR
  • Golden Gate
  • mRNA extraction

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 .

PCR Clean Up

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

Q5 PCR

Name	Amount
Q5 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.

Thermocycler settings:

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'

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

OneTaq colony PCR

Name	Amount
oneTaq 2x MM	6 µL
AD	5 µL
Primer forward	0.5 µL
Primer reverse	0.5 µL
Template Colony	1 tip
Total Volume	12 µL

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

Thermocycler settings:

Step	Temperature in °C	Time
1	94	3'
2	94	30''
3	variable - Primer annealing temperature	40''
4 → 2 30x	68	variable - 1' /kb
5	68	5'

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:

Substance	Amount
Empty Backbone 40 nM	1 µL
Backbone Insert(s) 40 nM	2 µL each
linear DNA Insert(s) 40 nM	4 µL each
GG Mix 20x	1 µL
GG Buffer 10x	2 µL
AD	variable
total volume	20 µL

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

Step	Temperature in °C	Time in minutes
1	37	2
2 → 1 10x	16	5
3	37	10
4	55	30
5	80	10
6	23	10

Golden Gate Mix is ready for transformation.