Team:BOKU-Vienna/Protocol
Protocol
Materials
LB medium
LB medium is a standard liquid medium for the cultivation of E. coli.
| Name | Amount |
|---|---|
| Peptone | 10 g |
| Yeast Extract | 5 g |
| NaCl | 5 g |
| RO-water | 1000 mL |
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.
| Name | Amount |
|---|---|
| Peptone | 10 g |
| Yeast Extract | 5 g |
| NaCl | 5 g |
| RO water | 986 mL |
| Agar | 14 g |
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.
| Name | Amount |
|---|---|
| Agarose | 5.4 g |
| 50x TAE buffer | 7.2 g |
| RO water | fill up to 360 g |
• 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
| 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.
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.
