Team:Sorbonne U Paris/Protocols

Protocols

On this page, you can find the protocols we used during de iGEM project. Feel free to have a look. If you have any questions, you can contact us at igemsorbonneuniv2018@gmail.com

Materials:
- Phusion DNA polymerase
- High Fidelity buffer (5X)
- Free dNTPs mix (10µM)
- Forward Primer (10µM)
- Reverse Primer (10µM)
- Sterile water
- Plasmid DNA
- PCR tubes (0.2 mL) for the PCR machine


Methods:
1. Choose the appropriate primers, for example use a software such as DNA strider
2. Prepare a pre-mix (without the primers and plasmid DNA) based on the reaction volume and the number of tubes

Components Volume for 1 reaction (in µL)
(total volume = 25 µL)
DNA template at 2 ng/µL 1
Phusion Enzyme* 0.25
HF Buffer (5X)* 5
dNTP* 0.5
Forward Primer 1.25
Reverse Primer 1.25
H20 up to 25 µL* 15.75

*in the master mix

3. Distribute 21.5 µL of master Mix per Tube
4. Add 1 µL of DNA and 1.25 µL of each Primer
5. Mix gently the reaction. If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
6. Put the PCR tubes to the thermocycler and parameter the cycles (take into account the melting temperature Tm for each
primer)
Annealing temperature (Ta) calculation: Ta = Tm – 5°C with Tm = 2°C x (A+T) + 4°C x (G+C) for primers less than 30 bases long.

Note: this approximate formula tends to overestimate the Tm value!

Step Temperature (°C) Time (minutes)
98 1
35 98 1
50 2
72 3
72 3
12 Hold

Table: Parameters of the thermocycler for our exepriments

Materials:
- Agarose Powder
- TAE buffer 10X (Tris-acetate 40 mM, EDTA 1 mM, pH 7.8)
- DNA ladder (NEB 1 log)
- Loading buffer 6X (NEB)
- Gel Tank
- Comb
- Balance
- Microwave oven
- Ethidium bromide 10mg/ml (EtBr)

Methods:

Preparation of agarose gel

% Gel Linear DNA size (Kb)
0.5 30 to 1.0
0.7 12 to 0.8
1.0 10 to 0.5
1.2 7 to 0.4
1.5 3 to 0.2
2.0 1 to 0.1

Electrophoresis gel resolution

Agarose gel x % (w/v) in 1X TAE buffer.
Chose the agarose concentration you need using the above table

According to the gel’s size, prepare the appropriate volume of solution (for exemple 50 mL for a small sized agarose gel)

1. Prepare 1% w/v solution of agarose powder in 1X TAE buffer (e.g. 1 g agarose powder in 100 mL TAE buffer) 2. Heat the solution in a microwave to dissolve agarose until the solution is limpid (usually 30-45 sec at max power, avoid boiling)
3. Cool the solution (not for too long or it will solidify). Avoid bubbles and vigorous stirring. You can allow hot agarose to cool in a water bath set at 50–55°C for 10 min. Swirl the flask occasionally to cool evenly.
4. Add Ethidium bromide EtBr solution(0.5mg/ml) to the cooled gel and pour into the gel tray
5. Cast the solution on an electrophoresis gel mould insert the comb and let the gel solidify at room temperature

Caution with hot agarose

Wear protective gloves when handling extremely hot agarose solution and heat the solution in several short intervals—do not let the solution boil for long periods as it may boil out of the flask or cause a loss to water vapor

Gel migration

1. Remove the comb
2. Immerse the gel with 1X migration buffer (TAE). Cover the gel with ~2–3 mm of buffer for best results.
3. Load 3 µL of the DNA ladder on a well
4. Load 15 µL DNA samples with the corresponding amount of DNA loading dye (DNA loading dye 1X)
Add 5 µL of 6X loading buffer to the 25 µL of each sample and load in the expected wells (respect chosen order) 5. Run the gel for 20 minutes at 100V

Caution with Ethidium Bromide

Ethidium Bromide is a sensitive fluorescent dye used to detect nucleic acids in agarose gels. It is a mutagen and probable carcinogen. It is toxic. Always wear gloves when working with ethidium bromide. Wipe the area with a damp cloth after the work with ethidium bromide. Also, while wearing gloves after handling ethidium bromide, be careful not to touch and thereby contaminate other surfaces. Be sure to wash hands with soap and warm water after working with ethidium bromide.

Gel revelation:

At the end of the electrophoresis migration, collect the Gel and take a picture of it using a gel reader, adjust the brightness if necessary.

The sample is mixed with Binding Buffer NTI and in case of a cut-out gel band, it is heated to dissolve the agarose. In the presence of chaotropic salt, the DNA is bound to the silica membrane of a NucleoSpin® Gel and PCR Clean-up Column. Contaminants are removed by simple washing steps with ethanolic Wash Buffer NT3. Finally, the pure DNA is eluted under low salt conditions with slightly alkaline Elution Buffer NE (5 mM Tris-HCl, pH 8.5).

Material:
- NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel)

Methods:
1. Spot on the gel the expected bands
2. Cut with scalpel on the gel the most precisely possible to extract the desired bands
3. Use the Gel DNA extraction kit to extract the DNA samples from the gel

DNA Gel extraction was performed according to the NucleoSpin® Gel and PCR Clean-up kit manufacturer’s instructions.

Materials:
- BbsI-HF (NEB)
- T4 ligase (NEB)
- Cutsmart buffer (NEB)
- ATP (10 mM solubilized in 0.1 M Tris-HCl, pH 7.9).



Methods:
To make our MoClo Assembly protocols, we used an excel table provided on the supplementary material from: “Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. Crozet P, et al. ACS Synth Biol. 2018”
For each reactions this excel table enabled us to calculate the appropriate volumes of water, DNA, cutsmart buffer, enzymes and additives (for example ATP), based on the DNA concentrations we had.


Reaction mix ul Master mix
BbsI-HF 1 2
T4 ligase 1 2
Buffer NEB cutsmart 2 4
ATP (10 mM) 2 4
Total volume 20 6 uL/tube

The parameters for the PCR thermocyclers following our digestion/ligation experiment were also based on the data from the article cited above, and whether we were performing a level 0 or level 1 cloning.


Step Temperature (°C) Time (minutes)
3 cycles 37 10
16 10
37 10
65 20
Hold 16 Indefinitely

Materials:
- Cut smart Buffer (10X)
- Enzyme BsaI-HF®v2 (NEB)
- Enzyme NdeI(NEB)
- DNA
- Sterile water

Methods:
1. Set up the reaction as follows

Component Volume for 1 reaction (in µL)
(total volume = 10 µL)
DNA 3
Cutsmart Buffer 10X 1 µL (1X)
BsaI or NdeI 0.2 µL (or 2 units)
Nuclease-free Water Up to 10 µL

Incubate at 37°C for 1 hou

Materials:
- FastDigest Buffer (FD) 10X (Thermofisher Scientific)
- BcII Enzyme (NEB)

Methods: 1. Set up the reaction as follows

Component Volume for 1 reaction (in µL)
(total volume = 10 µL)
DNA 3
FastDigest Buffer (10X) 1 µL (1X)
BclI Enzyme 0.2 µL (or 2 units)
Nuclease-free Water Up to 10 µL

2. Incubate at 37°C for 1 hour

Choose the appropriate bacterial strain based on your knowledge and your lab’s material. The strain of bacteria used for our experiments were chemically competent Escherichia coli DH5-alpha and 10-bêta kept at -80°C.

E. coli bacteria are transformed by the heat shock technique: chemically competent cells are incubated at 4°C with DNA for 20 min, then heat shocked at 42°C for 1 min and put back on ice for 2 min. Then the bacteria are cultivated in liquid LB medium supplemented with glucose for 1 hour at 37°C in order to reconstitute their weakened wall and to express the antibiotic resistance gene carried by the plasmids. The bacteria are then spread on the LB medium supplemented with Xgal and adapted antibiotic in order to select the bacteria that have been transformed by the plasmid.

Materials:
- E. coli 10-bêta (New England Biolabs® 10-beta Competent E. coli) or E. coli DH5-alpha
- ligation products (assay or negative control)
- LB medium
- Ice bucket filled with ice
- LB+ Xgal + spectinomycine solid medium
- Shaking incubator with modular temperature

Methods : 1. Take competent cells out of -80°C and thaw on ice (approximately 10 min).
2. Mix 4 µL of ligation products with 30 µL of E. coli 10-bêta bacteria (or 90 µL of E. coli DH5-alpha).
3. Incubate the competent cell/DNA mixture on ice (4°C) for 30 minutes.
4. Heat shock each transformation tube by placing the tube at 42°C for 55 seconds (E. coli 10-bêta bacteria) or 90 seconds (E. coli DH5-alpha).
5. Put the tubes back on ice (4°C) for 2 min.
6. Add then 700 µL of LB medium with glucose (without antibiotic) and leave to incubate 1 h at 37°C. Shake vigorously (250 rpm) or rotate
7. Spread 200 µL on plate containing LB + Xgal +spectinomycin

Incubate plates at 37°C overnight.
NB: Between the 2 and 4 steps, do not mix.

Material:
For plasmid extractions we performed minipreps using:
- NucleoSpin® Plasmid EasyPure (Macherey-Nagel),
- GeneJET Plasmid Miniprep Kit (Thermofisher Scientific)
- Nucleospin Plasmid Kit (Macherey-Nagel)

Methods:
Minipreps were carried out according to the manufacturer’s instructions.
After an overnight liquid culture, the plasmid DNA is extracted, based on the alkaline lysis technique of the bacterial cells. The DNA is purified by means of a silica membrane which selectively retains DNA and selectively ionic strength.

Material:
- Nanodrop 2000
- lint-free laboratory wipe
- Tip 2 μL
- Pipette 2 μL
- Samples

Methods:
The blank was made:
- using highly pure MilliQ water for ordered DNA sample
- using elution buffer from minipreps kits, for DNA previously extracted from minipreps
DNA concentration measurement was performed according to the NanoDrop 2000 spectrophotometer protocol.

1. Pipet 1 – 2 μL of sample directly onto the measurement pedestal
2. When the measurement is complete, raise the sampling arm and wipe the sample from both the upper and lower pedestals using a dry, lint-free laboratory wipe.

Note: Between measurements: Wipe the sample from both the upper and lower pedestals with a clean, dry, lint-free lab wipe.







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