Experiments
Here you can find the Experiments we conducted!
Materials
Resuspended DNA to be transformed
Competent Cells (50µl per sample)
LB Media
Petri dishes for selection
Protocol
Thaw competent cells on ice
Pipette 50µl of competent cells into 1.5ml tube
Pipette 1µl of resuspended DNA into 1.5ml tube
Incubate on ice for 30 minutes
Heat shock tubes at 42°C for 45 sec
Incubate on ice for 5 minutes
Add 950 µL of room temperature LB Media
Incubate at 37°C for 1 hours, shaking at 300rpm
Pipette 100µL of each transformation onto petri plates and plate them out
Incubate transformations overnight (14-18hr) at 37°C
Derived from the manual of our electroporation unit.
Materials
LB-medium
Ice
10% glycerol
DNA
SOC media
selection plates
Preparation of the cells
Inoculate 1 liter of LB-medium with 1/100 volume of a fresh overnight culture.
Grow cells at 37 °C with vigorous shaking to an ABS600 of approximately 0.5-0.7 (the best results are obtained with cells that are harvested at early- to mid-log phase; the appropriate cell density therefore depends on the strain and growth conditions).
To harvest, centrifuge cells in cold centrifuge bottles in a cold rotor at 4000 x gmax for 15 minutes.
Remove as much of the supernatant (medium) as possible. It is better to sacrifice the yield by pouring off a few cells than to leave any supernatant behind.
Gently resuspend the pellets in a total of 1 liter of ice-cold 10% glycerol taking care not to lyse them. Centrifuge as in step 3.
Resuspend in 0.5 liter of ice-cold 10% glycerol. Centrifuge as in step 3.
Resuspend in ~250 ml of ice-cold 10% glycerol. Centrifuge as in step 3.
Resuspend to a final volume of 3 to 4 ml in ice-cold 10% glycerol. The cell concentration should be about 1 - 3 x 1010 cells/ml.
This suspension may be frozen in aliquots on dry ice, and stored at -70 °C. The cells are good for at least 6 months under these conditions.
During these steps keep the cells as close to 0 °C as possible (in an ice/water bath).
Electro-transformation and Plating
Gently thaw the cells at room temperature and then immediately place them on ice. Remove the sterile cuvettes from their pouches and place them on ice. Place the white chamber slide on ice.
In a cold, 1.5 ml polypropylene tube, mix 40 μl of the cell suspension with 1 to 2 μl of DNA (DNA should be in a low ionic strength buffer such as TEc). Mix well and let sit on ice ~0.5 - 1 minute.
Set the E. coli Pulser apparatus to 2.50 kV when using the 0.2 cm cuvettes. Set it to 1.80 kV when using the 0.1 cm cuvettes. See Section 2 for operating instructions.
Transfer the mixture of cells and DNA to a cold electroporation cuvette, and shake the suspension to the bottom. Place the cuvette in a chilled safety chamber slide. Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber .
Pulse once.
Remove the cuvette from the chamber and immediately add 1 ml of SOCd medium to the cuvette and quickly but gently resuspend the cells with a pasteur pipette. (This rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants.)
Transfer the cell suspension to a 17 x 100 mm polypropylene tube and incubate at 37 °C for 1 hour. (Shaking the tubes at 225 rpm during this incubation may improve the recovery of transformants.)
Check and record the pulse parameters. The time constant should be close to 5 milliseconds. The field strength can be calculated as actual volts (kV) / cuvette gap (cm).
Plate on selective medium.
Materials
LB Media
DNA Miniprep Kit
Protocol
Transfer colony in question into 3mL of LB Media.
Incubate overnight at 37°C.
Pellet overnight culture via centrifugation at 16,000 x g (~13,000 RPM). Discard supernatant.
Resuspend pellet in 200 μl Plasmid Resuspension Buffer. Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
Lyse cells by adding 200 μl Plasmid Lysis Buffer. Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.
Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer. Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex!
Incubate for 2 minutes.
Clarify the lysate by spinning for 2–5 minutes at 16,000 x g.
Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Centrifuge for 1 minute.
Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
Transfer column to a clean 1.5 ml microfuge tube.
Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.
Check DNA concentration through Nanodrop.
Materials
Tryptone
Yeast Extract, bacteriological
NaCl (Sodium Chloride, Molec Biol grade)
Antibiotics stock, if required
Protocol
Weigh out the following ingredients into a 1L glass bottle:
5 g Tryptone
2.5 g Yeast Extract
5 g NaCl
Fill up with distilled water.
Mix by swirling.
Autoclave the solution.
Optional:
Led the Media cool down
Add 500 µL of antibody stock if required
Store the LB-Media at 4°C.
Materials
Tryptone
Yeast Extract, bacteriological
NaCl (Sodium Chloride, Molec Biol grade)
Agar, bacteriological
Antibiotics stock, if required
One use plastic petri dishes
Protocol
Weigh out the following ingredients into a 1L glass bottle:
5 g Tryptone
2.5 g Yeast Extract
5 g NaCl
7.5 g agar
Fill up with 500 mL distilled water.
Mix by swirling.
Autoclave the solution.
Optional
Let the Media cool down.
Add 500 µL of antibody stock if required.
Pour or pipette about 20 mL LB agar into each Petri dish under sterile conditions.
Place lids on Petri dishes and allow to cool and solidify (about 30-60 minutes).
Invert the plates to avoid dripping of moisture onto agar. Label with antibiotic(s) name and date.
Store plates at 4°C.
Materials
Solution TFB1:
Solution TFB2:
Protocol
Prepare a 10 mL liquid culture of the strain of interest
Incubate overnight
The following day, transfer 2 mL of this culture to a flask containing 200 mL LB medium and incubate until OD600 reaches 0.6.
Cool down the cells on ice for 10 min. The cells are pelleted in a centrifuge for 5 min at 4,500 rpm (4,000 × g) at 4°C. The cells are resuspended in 0.4 volume of ice-cold TFB1.
Repeat the centrifugation step. Resuspend the pellet in 1/25 volume of ice cold TFB2.
The cells are aliquoted 100 µL per tube and stored at −80°C.
Protocol
Prepare a Master mix
| Components |
10x Master Mix |
| Q5 Reaction bufffer |
50 µl |
| 10 mM dNTPs |
5 µl |
| Primer 1 |
12,5 µl |
| Primer 2 |
12,5 µl |
| Enhancer |
50 µl |
| Wasser |
107.5 µl |
Pipette 6 µL of the Master mix into each reaction tube
Add 4 µL of the Miniprep into each reaction tube.
Incubate for 1 h at 37°C
Separate the digest via gel electrophoresis
Protocol
Preheat the incubator to 29 °C.
Completely thaw the myTXTL Sigma 70 Master Mix. Keep on ice until use.
Directly before use, vortex the myTXTL Sigma 70 Master Mix for 2-3 seconds and briefly spin down.
Pipette 9 µL of the CFS mix into a reaction tube. Add 3 µL of DNA (optionally: positive & negative control) to each reaction tube.
Gently vortex the reaction mixture for 2-3 seconds and briefly centrifuge mixture.
Incubate the myTXTL reaction(s) for up to 16 h at 29 °C.
Stop the myTXTL reaction by placing the tube(s) on ice.
Place each reaction into a well for measurement.
Dilute each sample with 48 µL of distilled water (1:4 dilution)
Measure fluorescence Emission with the following parameters:
Extinction: 587nm
Emission: 610
Materials
Plasmids of interest
Cutsmart buffer
ATP
Restriction enzymes
T4 ligase
H20
Master mix preparation
| Component |
µL/sample |
µL Mastermix (5 reactions) |
| Insert 01 |
1 |
5 |
| Insert 02 |
1 |
5 |
| Plasmide Backbone 03 |
1 |
5 |
| Cutsmart buffer |
2 |
10 |
| ATP |
2 |
10 |
| restriction enzyme |
2 |
10 |
| T4 ligase |
0.25 |
1.25 |
| H20 |
9.75 |
48.25 |
| total volume |
20 |
|
The amount of water added can be variable based on the amount of different inserts used in cloning. In general the H2O should be used to bring the total volume up to 20 uL.
Thermocycler protocol GoldenGateshort:
40 cycles:
37°C - 1 min
16°C - 2.5 min
1 cycle:
37°C - 30 min
50°C - 5 min
80°C - 10 min
20°C - 2 min
Protocol
Prepare a master mix of all components except the new backbone
Pipette 19 uL of the master mix into separate eppies
Add 1 uL of DNA solution to the Mastermix.
Place the reaction tubes into the thermo cycler with the described programme
Protocol
Prepare a Master Mix:
| Components |
10x Master Mix |
| Q5 Reaction buffer |
50 µl |
| 10 mM dNTPs |
5 µl |
| Primer 1 |
12,5 µl |
| Primer 2 |
12,5 µl |
| Q5 High Fidelity DNA Polymerase |
2,5 µl |
| Wasser |
107.5 µl |
Pipette 24 µL of Master Mix into each reaction tube
Add 1 µL of DNA into each reaction tube
Place the reaction tubes into the thermocycler with one of the the following programmes:
Control success of amplification via gel electrophoresis
Protocol
A 10 mL liquid E. coli culture was prepared and incubated to an OD of around 0.7.
Once the OD is reached 1 mL of Phage stock is added to the culture.
Further incubate for around 2h. Measure the OD every 30 minutes. One should observe clearing of the culture and a drop of the optical density.
Spin down cells/cell debris using e.g. 10,000 x g spin 10 mins.
Take the supernatant and add solid PEG (2 g /L) and NaCl (2.9 g/L) assuming you started in LB or similar salt conc. Mix well. Centrifuge at 10,000 x g again for 10 min.
Take the supernatant and add more PEG (9 g /L) and NaCl (2.9 g / L) mix well.
Incubate the suspension on ice for 1h. Centrifuge at 15,000g for 45 minutes.
Resuspend in whatever volume you need.
Check titre
Based on Sayers & Eckstein, 1990.
Protocol
Pipette 100 µL of phage stock into a reaction tube
Add 100 µL of Phenol-Chloroform-Isomethylethanol
Spin 5 minutes at 16,000 rcf at RT
Repeat steps 2 & 3 twice.
Add 100 μl Chloroform.
Spin 5 minutes ar 16,000 rcf at RT
Transfer supernatant and add 10 μl 3M Na-Acetat.
Add 300 μl cooled 100% Ethanol.
Vortex and incubate on ice for 1 hour.
Spin for 30 minutes at 16,000 rcf.
Carefully discard the supernatant.
Elute the sample in 25 μl nf H2O.
Check the DNA concentration with Nanodrop, dilute if necessary.
If your browser does not support embedded PDFs you can open the protocols seperately: Plaque Assay_BOKU-Vienna & Infection Dose Assay_TUDelft
