Here you can find all our important protocols
Here you can find the protocols we used for our transformations
DNA
Distribution Kit
Competent cells
SOC Media
Resuspend DNA in selected wells in the Distribution Kit with 10μl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
Label 1.5mL tubes with part name or well location. Fill the lab ice bucket with ice, and pre-chill 1.5 mL tubes (one tube for each transformation, including your control).
Thaw competent cells on ice: This may take 10-20 min for a 260 μl stock.Dispose of unused competent cells. Do not refreeze unused thawed cells, asit will drastically reduce transformation efficiency.
Pipette 50 μL of competent cells into 1.5 mL tube: 50 μL in a 1.5 mL tube pertransformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 1.5 mL tube for your control.
Pipette 1 μL of resuspended DNA into 1.5 mL tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice. Mix gently, e.g. by moving the tube over a tube rack.
Close 1.5 mL tubes, incubate on ice for 15 min.
Heat shock tubes at 42 °C for 45 s. Ensure the bottoms of the tubes are submerged. Timing is critical.
Incubate on ice for 5 min: Return transformation tubes to ice bucket.
Pipette 800 μL prewarmed SOC media (37 °C) to each transformation.
Incubate at 37 °C for 1 ho at 300 rpm.
Centrifuge for 1 min at 4500 rpm, removement of the supernatant until 100 µL and suspend the pellet in remained medium. The cells are plated out onto (antibiotic) agar plates and incubated overnight at 37°C.
DNA
Distribution Kit
Competent cells
SOC Media
Resuspend DNA in selected wells in the Distribution Kit with 10μl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
Label 1.5mL tubes with part name or well location. Fill the lab ice bucket with ice, and pre-chill 1.5 mL tubes (one tube for each transformation, including your control).
Thaw competent cells on ice: This may take 10-20 min for a 260 μl stock.Dispose of unused competent cells. Do not refreeze unused thawed cells, asit will drastically reduce transformation efficiency.
Pipette 50 μL of competent cells into 1.5 mL tube: 50 μL in a 1.5 mL tube pertransformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 1.5 mL tube for your control.
Pipette 1 μL of resuspended DNA into 1.5 mL tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice. Mix gently, e.g. by moving the tube over a tube rack.
Close 1.5 mL tubes, incubate on ice for 15 min.
Heat shock tubes at 42 °C for 45 s. Ensure the bottoms of the tubes are submerged. Timing is critical.
Incubate on ice for 5 min: Return transformation tubes to ice bucket.
Pipette 800 μL prewarmed SOC media (37 °C) to each transformation.
Incubate at 37 °C for 1 ho at 300 rpm.
Centrifuge for 1 min at 4500 rpm, removement of the supernatant until 100 µL and suspend the pellet in remained medium. The cells are plated out onto (antibiotic) agar plates and incubated overnight at 37°C.
Template DNA
Forward Primer/ Reverse Primer
Buffer
10 mM dNTPs
Taq Polymerase
LB-Agar plate
Prepare the mastermix with the following reagents:
|
Reagents |
Volume |
|
Buffer |
20 µL |
|
10 mM dNTPs |
2 µL |
|
Taq Polymerase |
1 µL |
|
Water |
65 µL |
Add 0.5 µL of each primer (forward and reverse primer) to 8.8 µL of Mastermix.
Add 0.2 µL of ORI to the corresponding tubes.
Pick three colonies from each plate.
Inoculate the labeled spot on an LB-Agar plate.
Shortly dip the rest of the colony into the PCR mix, then resuspend in LB-Medium with Camp for overnight cultures.
Transfer tube to a thermocycler with the following program:
|
Temperature (°C) |
Time (mm:ss) |
Cycle |
|
98 |
10:00 |
|
|
95 |
00:30 |
25 |
|
xx |
00:30 |
25 |
|
72 |
xx |
25 |
|
72 |
5:00 |
agarose gel with DNA from gelelecktrophoresis
Membrane Binding Solution
SV Minicolumn
Membrane Wash Solution
Nuclease-free water
Slice DNA out of the agarose gel and weigh the gel sclice.
Add Membrane Binding Solution at a ratio of 10 µL/10 mg of agarose gel slice.
Insert SV Minicolumn into the tube.
Incubate at 60 °C for 10 minutes or until the slice is completely dissolved and vortex every three minutes
Centrifuge at 16,000 x g for 1 minute, then discard the flowthrough and reinsert SV Minicolumn.
Add 700 µL Membrane Wash Solution (ethanol added). Centrifuge at 16,000 x g for 1 minute, then discard flowthrough and reinsert SV Minicolumn.
Add 400 µL Membrane Wash Solution. Centrifuge at 16,000 x g for 5 minutes, then discard flowthrough and reinsert SV Minicolumn.
Empty the tube and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open to allow evaporation of any residual ethanol.
Transfer Minicolumn to clean 1.5 ml microcentrifuge tube.
Add 50 µL of Nuclease-Free Water to Minicolumn and incubate at room temperature for 1 minute. Centrifuge at 16,000 x g for 1 minute.
Discard Minicolumn and store DNA at -20°C.
15 mL culture tube
LB medium
1000X antibiotic stock solution
Plate with colonies
Micropipette tips
Add 2-5 mLLB medium into a culture tube and add 1 uL of antibiotic stock solution per 1 mL of medium.
Pick one colony using a micropipette tip and eject it into the culture tube.
Incubate overnight at 37°C and 150 - 250 RPM.
Template DNA with part
Q5 High-Fidelity DNA-Polymerase
5XQ5 Reaction Buffer
10 mM dNTPs
10 uMFw-Primer
10 uMRw-Primer
Nuclease-free water
DNA dilution to 10 ng/µL
Primer dilution to 100 µM
Setup the PCR reaction mixes with each following the scheme:
|
Component |
50 uL Volume (same ratios for the 20 uL reactions) |
Final concentration |
|
5x Q5 Reaction Buffer |
10 |
1x |
|
10 mM dNTPs |
1 |
200 uM |
|
10 uM Fw |
2,5 |
0,5 uM |
|
10 uM Rv |
2,5 |
0,5 uM |
|
Template DNA (10 ng/uL) |
1 |
10 ng / 50 uL reaction mix |
|
Q5 High-Fidelity DNA-Polymerase |
0,5 |
0,02 U/uL |
|
Nuclease-free water |
32,5 |
|
Run PCR following the scheme:
|
step |
temperature |
time |
|
Initial denaturation |
98°C |
30 seconds |
|
35 cycles |
98°C 69°C 72°C |
10 seconds 30 seconds 30 seconds/kb see |
|
Final Extension |
72°C |
2 minutes |
|
Hold |
4 °C |
Agarose
1 x TAE buffer
Gel chamber
DNA
6 x loading dye
DNA ladder
SYBR Green
Camera
Completely dissolve agarose in an appropriate concentration (e.g. 1 % ) in TAE buffer by heating.
Pour the solution in a gel chamber using a fitting comb and wait for the gel to fully polymerize (up to an hour).
Mix at least 10 µL of the PCR products with loading dye and 1:10 SYBR Green.
Load the gel with the samples and the DNA ladder.
Run the samples at 100-120 V for 30-45 minutes.
Analyze and image the gel under a camera with an UV illuminator.
Cells
Centrifuge
LB-Medium
Ca/Glycerol buffer (60 mM CaCl2, 10 mM PIPES, 150 mL glycerol, fill water up to 1 L, pH=7.0)
Liquid nitrogen
Grow the cells at 37 °C till OD= 0.2/0.4.
Chill the culture on ice for 5 min.
Collect cells by centrifugation at 6000 rpm at 4 °C for 10 min.
Gently resuspend the cells from 500 mL LB in 40 mL cold Ca/glycerol buffer on ice.
Incubate the cells on ice for 5~30 min.
Repeat step 3 and 4.
Incubate the resuspended cells in Ca/glycerol buffer on ice for 30 min (the longer, the better).
Collect celly by centrifugation at 6000 rpm at 4 °C for 10 min.
Resuspend cells from 500 mL LB in 6 mL Ca/glycerol buffer.
Aliquote 150 µL/tube.
Freeze in liquid nitrogen and store at -80°C.
Bacterial culture
Centrifuge
Buffer LYSIS LP
Buffer RESUSPENSION
Buffer NEUTRALIZATION
Buffer Wash PA
Buffer Wash PB
Buffer Elution EP
Collection Tube with Mini Filter
Transfer the culture into 2 mL Eppis.
Centrifuge at maximum speed for 1 min to pellet the cells and remove the supernatant completely and discard.
Resuspend the pellet in 250 µL Buffer RESUSPENSION and mix by vortexing or pipetting up and down.
Add 250 µL Buffer LYSIS LP and mix by inverting the tube seven times.
Incubate at 20 °C for 5 min. Invert the tube few more times during incubation for improved lysis.
Add 350 µL Buffer NEUTRALIZATION and mix gently.
Centrifuge at maximum speed for 8 min to precipitate proteins and large chromosomal DNA.
Transfer the clarified supernatant to Mini Filter (orange) placed in a Collection Tube.
Centrifuge at 12000 rpm for 1 min, discard the filtrate and re-use the Collection Tube.
Add 500 µL Buffer WASH PA to the Mini Filter.
Centrifuge at 12000 rpm for 1 min, discard the filtrate and re-use the Collection Tube.
Add 700 µL Buffer WASH PB.
Centrifuge at 12000 rpm for 1 min, discard the filtrate and re-use the Collection Tube.
Centrifuge at maximum speed for 2 min to remove residual ethanol,
Place the Mini Filter in a new Elution Tube and add 20 µL water to the center of the Mini Filter.
Incubate at 20 °C for 1 min.
Centrifuge at 8000 rpm for 1 min and discard the Mini Filter.
Purified plasmid DNA in the Elution Tube can be used immediately.
Store the DNA at -20 °C.
96 well microplate
Bacteria culture expressing a reporter detectable at a specific wavelength
LB media
Microplate reader
Pipette 300 μL of each culture in the top row of the 96 well plate (under the clean bench). Pipette the same volume of LB media into an additional top row well.
Pipette 200 μL of LB media into the rows beneath for each dilution
Pipette the volume of culture from the top row into the row beneath corresponding to the desired dilution (e.g. 50 μL for a 1:5 dilution) and resuspend 2-3 times
Repeat Step 3 for each of the following dilutions
Discard 50 μL of the last dilution
Place the lid on the 96 well plate and place it in the microplate reader
Use an excitation wavelength and detect an emission wavelength corresponding to the used reporter