Figure 1Diagram of procedure for bacterial transformation protocol2
Materials
15 μl of E.coli competent cells (per sample)
1.5 μl of the plasmid of interest
1.5 μl of water
100 μl of SOC medium (per sample)
LB-agar with Kanamycin plates
Setup and protocol
In case the plasmid is dried, add 40 μl of water in the 4 μg of plasmid to obtain a plasmid concentration of 100 ng/μl. Do not forget to label the new concentration.
Thaw frozen Competent Cells on ice until just thawed. For the rest of the experiment, it is important to work with fire to avoid any contamination AND keep everything on ice.
Add 1.5 μl of plasmid or water per 15 μl tube of competent cells. Quickly flick the tube several times.
Return the tubes to ice for 10 minutes. The protocol indicates to put the tube on ice for 30 minutes.
Heat-shock the cells for 30 seconds at 42°C without shaking.
Immediately place the tubes on ice for 2 minutes.
Add 100 μl of room temperature SOC medium to each transformation reaction. Incubate for 30 minutes at 37°C with shaking (300rpm).
Incubate for 30 minutes at 37°C with shaking (300rpm).
Colony picking
Figure 2Diagram of procedure for colony picking protocol
< 2
Materials
Pipette girl
Micropipettes
LB medium + Kanamycin
100 μl of SOC medium (per sample)
Colony plates
15 ml tubes
Protocol
Into a 15ml tube, put 10ml of sterile LB medium and 100μl of Kanamycin (or put 10ml of already prepared LB-Kana 1X).
Using the tip of a sterile pipette, scoop up a colony and add it into the tube. Be careful to avoid satellite colonies, as they could be contaminated by non-resistant bacteria.
Place the tube in the shaker at 200 rpm and at 37°C for 12h, and open the cap to allow oxygen renewal as well as avoid condensation. The shaker needs to be equilibrated.
Prepaid Microsynth tubes + labels for E.coli NightSeq
Protocol
Transfer 10 μl of the bacterial culture in a tube and label it appropriately according to Microsynth procedure.
Register on the Microsynth website, choose Sanger sequencing- Single Tube Sequencing - E.coli NightSeq fill the order form and send the samples to Microsynth.
Centrifuge 1.5ml of bacterial culture for 30 seconds at maximum speed in a microcentrifuge. Discard the supernatant.
Add an additional 1.5ml of bacterial culture to the same tube and repeat Step 1.
Add 600μl of TE buffer or water to the cell pellet, and resuspend completely. (Alternative step : Add 600μl of bacterial culture to a 1.5ml microcentrifuge tube if smaller culture volume.)
Add 100μl of Cell Lysis Buffer (Blue), and mix by inverting the tube 6 times.
Add 350μl of cold (4–8°C) Neutralization Solution, and mix thoroughly by inverting.
Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
Transfer the supernatant (~900μl, not more) to a PureYieldTM Minicolumn without disturbing the cell debris pellet.
Place the minicolumn into a Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 seconds.
Discard the flowthrough, and place the minicolumn into the same Collection Tube.
Add 200μl of Endotoxin Removal Wash (ERB) to the minicolumn. Centrifuge at maximum speed inside a microcentrifuge during fifteen seconds.
Discard flowthrough.
Add 400μl of Column Wash Solution (CWC) to the minicolumn. Centrifuge at maximum speed inside a microcentrifuge during thirty seconds.
Transfer the minicolumn to a clean 1.5ml microcentrifuge tube (label the tube), then add 30μl of Elution Buffer directly to the minicolumn matrix. Let stand for 1 minute at room temperature.
Centrifuge for 15 seconds to elute the plasmid DNA. Keep the 1.5ml tube containing eluted DNA and trash the column.
Determine concentration and purity (A260/A280) of the DNA sample with NanoDrop.
Big bacterial culture (two different colonies from the same plate)
LB medium + Kanamycin (1X)
15 ml falcon tubes
Micropipettes
Pipetboy
Protocol
Turn on the flame to create a sterile environment where we can safely manipulate bacteria and the LB medium.
Pipette 5 mL of LB medium + Kanamycin into the 15mL falcon tube.
Add 50 uL of bacteria.
Put the falcon tube on a shaker at 30°C (better if it is 37°C) overnight.
Measure the OD600 of the bacterial culture by putting 1 mL of culture in a cuvette. If it is between 0.4 and 0.6 then we can directly do the IPTG induction (it shows that the bacteria are in the exponential growth phase).
Pipette 2 mL of bacterial culture with appropriate OD in a 5mL tube.
Add the necessary amount of IPTG solution in the corresponding sample.
Figure 6Diagram of procedure for MagneHis Purification kit2
Protocol
Bacterial cell lysis
Determine the OD600 for the fresh bacterial culture. The cell culture should have a final OD600
< 6.0 for efficient processing.
Centrifuge 1ml of bacterial culture at 10,000 x g for 2 minutes in a microcentrifuge. Remove the supernatant completely. We used a paper to get rid of the final supernatant residu. No pipetting is needed.
For every 1 OD600, dilute 10μl of FastBreakTM Cell Lysis Reagent, 10X, to 100 μl (1X) by adding 90 μl of double-distilled water. Do not process more than 1 OD600 of culture per 100 μl of 1X FastBreakTM Cell Lysis Reagent.
Resuspend the cell pellet in 1X FastBreakTM Cell Lysis Reagent. After this step, always vortex and put the sample on ice after pipetting !
Resuspend lyophilized DNase 1 as indicated on the vial if not already done (80 μl of water added to the DNase 1), and add 1 μl to the lysed bacterial culture. Store the resuspended DNase 1 in aliquots at -20°c for long term or at 4°c for up to one week.
Incubate with shaking for 10-20 minutes at room temperature on a rotary mixer or shaking platform.
Purification of Polyhistidine proteins for 1 ml of bacterial culture
DO NOT FORGET TO LABEL THE TUBES
Vortex the MagneHisTM Ni-Particles to a uniform suspension. It is very important to vortex the beads because they always clump together.
Add 30μl of MagneHisTM Ni-Particles either to cell pellet resuspended in 1X FastBreakTM Cell Lysis Reagent. Note: You may need to increase the amount of MagneHisTM Ni-Particles used for high-expressing proteins.
Invert tube to mix (approximately 10 times), and incubate for 2 minutes at room temperature. Make sure the MagneHisTM Ni-Particles are well mixed.
Place the tube in the appropriate magnetic stand for approximately 30 seconds to capture the MagneHisTM Ni-Particles. Using a pipette, carefully remove the supernatant, and put it in a new 1.5 ml tube.
Remove the tube from the magnetic stand. Add 150μl of MagneHisTM Binding/Wash Buffer to the MagneHisTM Ni-Particles. Resuspend up and down by pipetting the beads with the buffer.
Place the tube in the appropriate magnetic stand for approximately 30 seconds to capture the MagneHisTM Ni-Particles. Using a pipette, carefully remove the supernatant, and put it in a new 1.5 ml tube.
Repeat the wash step 2 times for a total of 3 washes. Always resuspend up and down by pipetting the beads with the buffer.
Remove the tube from the magnetic stand. Add 100μl of MagneHisTM Elution Buffer, and pipet to mix (or vortex).
Incubate for 1–2 minutes at room temperature. Place in a magnetic stand to capture the MagneHisTM Ni-Particles. Using a pipette, remove the supernatant containing the purified protein and put it in a new 1.5 ml tube.
Analyze the samples by SDS-PAGE or by functional assay.
Protein purification PTPSP
Protein purification protocol, as realised at the Protein Production and Structure Core Facility, at EPFL
Figure 7Diagram of procedure for Protein purification from PTPSP2
Materials
2.5M imidazole stock solution
5M NaCl solution
Protease inhibitor tablet
Turbonuclease
Sonicator
1M HEPES
HisPur Ni-NTA beads in 20% ethanol
Disposable plastic column
Centrifuge going to 20 000 xg
0.30 μm filters
Tube of bacterial culture induced with IPTG
Protocol
Prepare buffers
Wash buffer A (1 l)
300 mM NaCl
20 mM HEPES pH 7.5
20 mM imidazole (for non specific binding)
Elution Buffer B (250 ml)
300 mM NaCl
20 mM HEPES pH 7.5
500 mM imidazole
We use stocks of 5M Nacl, 1M HEPES pH 7.5 and 2.5M imidazole pH 7.5
Preparation of our sample
Defrost your tube of pellet in water, or obtain a cell pellet by centrifugation at 5000xg for 10min at 4°c.
Dilute the pellet with the wash buffer (ideally we need big volumes). We resuspent our cell pellet into 20mL. Bigger the volume is, the better. However, be careful to not completely drown the pellet with the wash buffer.
Addition of the following can help :
Add 1 protease inhibitor tablet. As previously, just approximate and don’t pipette it. Protease inhibitor protects the proteins sensitive to degradation and inhibit their destruction.
Add 5 ul of turbonuclease. Pipette the precise volume. Turbonuclease cleaves DNA to avoid the formation of jelly that is due to the presence of undestroyed DNA or RNA. Therefore, it ensures a better fluidity for our sample.
Mix well and vortex quickly, it makes the sample more homogenous. We can be harsh to cells without fragilising them. WARNING : After this step do not vigorously shake your sample !After cell lysis, proteins are not within the protective environment of the cell.
Put the cultures on a shaker at 4°c for 20min.
Lyse cells with a sonicator at 70% power, 10 secs on and 10 secs off pulse cycle over 2.5 minutes (in total, set the machine for 5 minutes). The samples will get hot. Then, put the sample on ice. Place the sample on ice in a beaker. Wash the tip with alcohol and water before and after. Sonication uses sound waves to explode the cells.
Centrifuge the lysed cells (lysate) at 20 000 x g for 30 minutes (1 hour or 2 hours could still be fine). For the balances for the centrifuge, take a flask and fill it approximately with the same volume of the sample with water. To open the machine, push the cover and pull it out. Stay next to the machine till it shows that it has reached the wanted speed. The sample should be less viscous after the centrifugation.
Resin preparation
If needed, pour out a bit of ethanol from the Ni-NTA beads in solution.
Equilibrate 2-5 mL Ni-NTA beads into the wash buffer (this removes the 20% ethanol in which the beads are stored), 2 mL for low expressing protein constructs, 5 mL for high expressors.
Measure out 2-5 mL of settled beads (we measured 25 mL of the beads in ethanol which corresponded to 5 mL of settled beads)
Empty (as much as possible) the ethanol without letting go of beads.
Repeat 3 times :
Add the wash buffer to around 40-50 mL, mix a bit.
Centrifuge the tube at 500g for 5 minutes (do not forget to balance the centrifuge with a tube of water).
Empty (as much as possible) the supernatant.
Incubation of lysate with Ni-NTA resin
When the centrifuge is finished, carefully transfer the supernatant into a beaker on ice. Keep some in the tube for the SDS-PAGE.
With a syringe, aspirate the supernatant CAREFUL NOT TO MAKE BUBBLES. If you are using a machine : filter the buffers using a 0.45 μm filter.
Add imidazole to the filtered supernatant to 25 mM. This reduces non-specific binding of endogenous His-rich E.coli proteins.
Decant the excess liquid from the washed beads.
Add the supernatant to the beads.
Put the beads with the supernatant rotating overnight at 4°c.
Purification
Setup a stand with a plastic disposable column.
Transfer the beads to a disposable plastic column.
Wash the beads in sequential order to remove loosely bound proteins before elution. The washes contain increasing amounts of imidazole to remove unbound, unspecific proteins from the resin. Do not forget to label everything and to keep everything to know where the proteins are. Keep all your fractions on ice.
Wash 1 - 20 mM imidazole - 30 mL
Wash 2 - 50 mM imidazole - 30 mL
Elute 1 - 250 mM imidazole - 30 mL
Elute 2 - 500 mM imidazole - 30 mL
If needed:
Elute 3 - 1 M imidazole - 20 mL
Elute 4 - 2.5 M imidazole - 10 mL
Elute 5 - 5 M imidazole - 10 mL
We had to do more elution steps than planned because the control (GFP protein) which contains both the CBD domains and the mSA didn’t eluted at the normal elution concentration. In what fraction the protein elutes depends on the size of the His-tag, where the tag is (N or C) and the 3D context of the target (is the tag accessible to the resin).
Concentration
Concentrate the protein using a filter of 30kDa.
Try to remove the imidazole excess by puting the concentrated proteins under dialysis. The dialysis buffer was composed of 300mM NaCl and 20 mM HEPES.
To see if there are protein aggregations, we centrifuged the result after dialysis. If a pellet appears, it means that there is an aggregation of protein which may mean that the proteins are not functional anymore.
Finally we measure the concentration of the protein after dialysis
Sample Buffer (2X) has to represent half of the final SDS sample volume (dilution factor of 2). Volumes are provided for a 20-μL sample size. Scale volumes proportionally for larger sample sizes. insert table
Heat denaturing samples at 85°C for 2 minutes. This step will facilitate the running of our samples.
Prepare buffer
Add 50 ml of NuPAGE MES SDS Running Buffer NP0002 (20X) and 950 ml of distilled water to obtain a 1X Running Buffer.
Prepare the gel
Open the gel packaging and remove the white tape near the bottom of the gel cassettes. It is very important to close the electric circuit !
Place the gels in the mini gel tank.
Fill the tank chambers with 1X Running buffer below the fill line (around 400 ml, stop before reaching the wells for better visibility while loading).
Remove the comb, and rinse the gel wells three times using 1X Running Buffer, try to remove the bubbles from the wells.
Load and run the gel
Load the samples in the wells of the gel
Load 3 μL of the protein ladder
Load 20 μL of the samples (or more, max 60 μL for 10-well gels and 35 μL for 15-well gels)
Fill the chamber with 1X Running buffer up to the fill line (also on the back of the gel)
Place the lid on the tank and connect the cables with generator
Run for 1 hour at 100 Volts
Recovery of the gel
Remove the gel from the tank and carefully cut the lower edge.
Crack the frame of the gel with a spatula. Put a little bit of buffer on the spatula and do not slide over the gel but rather cut it sequentially so it doesn’t rip the gel.
Put the gel in a box containing the running buffer, and carefully remove the buffer from the box.
Add the blue Protein Stain on top of the gel to cover it completely, and wait for ~15 min (up to 1 hour) on a rotary shaker at 10 rpm. This should allow the visualization of the protein.
Remove the stain, add distilled water to wash the stain and wait around 30min on a rotary shaker. Repeat this step if the protein is still not visible (incubation O/N possible).
6X anti His-Tag antibody conjugated to Alexa488 fluorophore
iBlot (Invitrogen, IB301002)
Wash buffer : PBS-Tween
Blocking buffer : 5% milk
Running buffer : 1X MOPS
SDS-PAGE gels not stained with Protein stain
Ponceau
Illuminator
Protocol
1X MOPS running buffer (500 ml)
1X MOPS from 20X MOPS (NuPage Ref:NP0001) using demineralized H20.
PBS-Tween wash buffer (1l)
1X PBS (10X PBS from facility in demineralized H2O).
0.1% Tween 20 (viscous and located under chemical hood).
5% milk blocking buffer (20 ml per membrane)
5% Milk Powder (weight/volume) in PBS-Tween. Keep on ice for antibodies, use around 7.5 mL per membrane.
Gel preparation and transfer
Give the gel a quick wash with the running buffer.
Soak the filter paper for the iBlot in the running buffer and follow the instructions of the iBlot manual.
Do the transfer using the iBlot system for 15 minutes on program 4 (15V).
Washes
Transfer the membrane to an appropriate container.
If the gel was not stained :
Check your transfer by covering the membrane in Ponceau and incubating briefly at room temperature.
Wash the membrane with dH2O until Ponceau/ the stain used is gone (using 1X PBS buffer is faster).
Blocking and antibody incubation
Incubate the membrane in 5% Milk on a rocking platform for 30 minutes (blocking).
Add the conjugated antibody to the 5% Milk using the company's provided instructions and keep on ice.
Incubate the membrane at least 1h at room temperature with the conjugated antibody in milk (10 mL for a large iBlot membrane) or overnight at 4°C with a parafilm cover to avoid evaporation.
Washes
Wash 3x quickly with PBS-Tween (add and discard right away).
Wash 3x for at least 10 minutes on a rocking platform at room temperature with PBS-Tween.
If you have a secondary antibody, repeat the last 3 steps for the secondary antibody.
Blotting (if not using HRP conjugated antibodies)
Put the membrane in an Illuminator and select the corresponding wavelength for the molecule the (secondary) antibody is conjugated to.
Observe any fluorescent band and save a picture of the membrane.
Drop-Plot
Materials
mSA-GFP-CBD (03a) protein purified
GFP (control) protein purified re-diluted in the wash Buffer A (300mM NaCl + 20mM Hepes) to have the same concentration as mSA-GFP-CBD (03a)
Nitrocellulose membrane
PBS
Petri dishes
Protocol
Cut a square of nitrocellulose membrane and put it in a petri dish.
Add 100 ul of both proteins (GFP control and mSA-GFP-CBD) on top of the nitrocellulose membrane.
Quickly see under UV light if you can observe fluorescence. If not, add more proteins (go with steps of 100 ul).
Observe more precisely the fluorescence under a fluorescence microscope and take an image.
Wait 15-20 min and wash the membrane with 8ml of PBS by shaking gently for 5 min.
Observe the potential fluorescence from the GFP proteins (GFP and mSA-GFP-CBD) under a fluorescence microscope and take an image.
Repeat the two previous steps two times (to perform 3 PBS-washes).
Wash the membrane with 8ml of Wash1 solution (pH5) by shaking gently for 5 min.
Observe the potential fluorescence from the GFP proteins (GFP and mSA-GFP-CBD) under a fluorescence microscope and take an image.
Repeat the two previous steps two times (first with Wash2 solution then with Wash3 solution).
Template DNA : the plasmids we want to modify (01a, 01b and 03a)
Platinum™ SuperFi™ DNA Polymerase
PCR machine
Materials - Method 2 (Marine’s lab)
Nuclease-free Water
DreamTaq™ Green PCR Master Mix
Forward Primer (10 μM stock)
Reverse Primer (10 μM stock)
DMSO
DNA : the plasmids we want to modify
PCR machine
Protocol
Preparation of PCR Mix Reaction
Method 1, using the reagents in our lab:
Prepare a 50 μl reaction in small PCR tubes (200 μl) as follows (add reagents in the same order as the one in the table) :
Table 1Reagents (Method 1)
Components
50 μl Reaction
Nuclease-free Water
up to 50 μl → 32.5 μl
5X SuperFi™ Buffer
10 μl
Forward Primer (10 μM stock)
2.5 μl (0.5 μM final concentration)
Reverse Primer (10 μM stock)
2.5 μl (0.5 μM final concentration)
dNTP mix
1 μl
Template DNA (100 ng/μl)
100 ng → 1 μl
Platinum™ SuperFi™ DNA Polymerase
0.5 μl
Mix well by pipetting up and down. Avoid making bubbles. Centrifuge shortly if needed.
Method 2, using the reagents in Marine's lab:
Prepare a 50 μL reaction in small PCR tubes (200 μL) as follows (add reagents in the same order as the one in the table):
Table 2Reagents (Method 2)
Components
50 μl Reaction
Nuclease-free Water
17.5 μl
Template DNA
1 μl
DMSO
1.5 μl
Reverse Primer (10 μM stock)
2.5 μl (0.5 μM final concentration)
Forward Primer (10 μM stock)
2.5 μl (0.5 μM final concentration)
Reverse Primer (10 μM stock)
2.5 μl (0.5 μM final concentration)
DreamTaq™ Green PCR Master Mix
25 μl
Mix well by pipetting up and down. Avoid making bubbles. Centrifuge shortly if needed.
Set-up of PCR machine
3-step protocol for inferior to 10 kb
Table 3PCR steps
Stage
Temperature
Time
Number of cycle
Initial denaturation
98°C
1 min
1X
Denaturation
98°C
10 sec
35X
Annealing
63.6°C → Depends on primers1
30 sec
35X
Extension
72°C
20-30 sec/kb → 3 min2
35X
Final extension
72°C
5 min
1X
Final extension
4°C
Hold
1X
1 We chose the annealing temperature of the primers using the Tm Calculator : 63.6°C. 2 For the time of the extension step, count roughly 20-30 sec/kb of the template DNA to amplify.
The biggest template we want to amplify (01a) is ~7600 bp, by taking 20 sec/kb we obtain 150 sec.
Agarose gel preparation (1% agarose) while PCR is running
Dilute 100 ml of TAE Buffer 10X into 900 ml of distilled water to make TAE Buffer 1X
Prepare 60 ml of 1% agarose gel by weighing 0.6 g of agarose powder and adding 60 ml of TAE 1X (for a small gel).
Heat the bottle of agarose gel in the microwave to dilute the agarose and obtain a homogeneous solution. DO NOT CLOSE THE BOTTLE WHEN HEATING IN THE MICROWAVE. Check that it doesn’t explode every ~30 sec, shake the bottle to mix.
Add 6 μl of SYBR Safe DNA Gel Stain (10 000X) in the hot melted agarose gel.
Let cool down for about 5 min.
Prepare the electrophoresis chamber to cast the gel, place the comb with the appropriate number of wells
Cast the gel inside the chamber and let cool for about 1 hour.
Samples preparation and loading on the agarose gel
For Method 1, using HESTIA's reagent, add 5 μl DNA 6X loading dye to 25 μl of PCR sample, mix by pipetting.
For Method 2, using reagents of Marine’s lab there is no need to add loading dye since it is integrated in the PCR master mix.
Add some TAE Buffer 1X on the gel to fill the wells before loading.
Load 10 μl of DNA ladder (SmartLadder from Eurogentec)
Load 30 μl of each samples on the gel
Fill the chamber with TAE Buffer 1X
Agarose gel electrophoresis
Assemble the electrophoresis unit. Make sure the anode cable (black) is plugged in the loaded DNA side (negative charges with negative charges for migration effect).
Run at 64 V for about 1h. Check at the image illuminator, run until the separation is good enough
Once the DNA separation is satisfying, remove the gel from the chamber and take a good image at the illuminator
Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5ml microcentrifuge tube.
Add 10μl Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 50–65°C until the gel slice is completely dissolved.
Binding of DNA
Insert SV Minicolumn into Collection Tube.
Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
Washing
Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
Repeat Step 4 with 500μl Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes.
Empty the Collection Tube and re-centrifuge the column assembly for 1 minute with the microcentrifuge lid open to allow evaporation of any residual ethanol.
Elution
Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube.
Add 50μl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute.
Mix well by pipetting up and down. Incubate at room temperature (25°C) for 5 minutes. Place on ice or store at -20°C.
Quick ligation
Materials
Purified plasmid DNA
Quick ligation Kit
Quick ligase reaction buffer (2X)
Quick ligase
Nuclear Free Water
Protocol
Set up the following reaction (table below) in a microcentrifuge tube on ice. *(Quick Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.)
Gently mix the reaction by pipetting up and down and microfuge briefly.
Incubate at room temperature (25°C) for 5 minutes to 2 hours.
Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells. Alternatively, Store at -20°C.
Do not heat inactivate – heat inactivation dramatically reduces transformation efficiency.
Table 2Summary of quantities for quick ligation protocol
Component
20 μl reaction
Quick Ligase Reaction Buffer (2X)*
10 μl
Vector DNA (4kb)
50 ng (0.020pmol)
Insert DNA (1kb)
37.5 ng (0.060 pmol)
Nuclease-free Water
to 20 μl
Quick Ligase
1 μl
Restriction Digestion and Agarose Gel Electrophoresis9
Materials
Plasmid DNA
Digestion buffers (10X rCutSmart Buffer and NEBuffer r3.1 for NcoI)
Nuclease free water
Restriction enzymes (SalI-HF, PmeI, NcoI, AscI and BstBI)
DNA 6X loading dye
DNA ladder (1 kb GeneRuler)
Agarose
TAE Buffer 1X (dilution from 10X stock)
Agarose gel electrophoresis tank
Generator
Protocol
Sample preparation
Set up the reaction as following
Table 3Version 1 of components quantities for Restriction digestion
Components
50 μl Reaction
DNA
1 μg (can vary)
10X rCutSmart Buffer
5 μl
RE
1 μl per RE
Nuclease free water
Up to 50 μl
Table 4Version 2 of components quantities for Restriction digestion
Components
20 μl Reaction
DNA
100 ng (can vary)
10X rCutSmart Buffer (NEBuffer r3.1 when NcoI is used)
2 μl
RE
0.5 μl per RE
Nuclease free water
Up to 20 μl
Digestion reaction
Digestion reaction
Incubate at 37°C for at least 2-3 hours to ensure digestion (digestion overnight is even better).
Agarose gel preparation (1% agarose)
Dilute 100 ml of TAE Buffer 10X into 900 ml of distilled water to make TAE Buffer 1X
Prepare 130 ml of 1% agarose gel by weighing 1.3 g of agarose powder and adding 130 ml of TAE 1X (130ml for a big gel, 60 ml enough for a small gel).
Heat the bottle of agarose gel in the microwave to dilute the agarose and obtain a homogeneous solution. DO NOT CLOSE THE BOTTLE WHEN HEATING IN THE MICROWAVE. Check that it doesn’t explode every ~30 sec, shake the bottle to mix.
Add 13 μl of SYBR Safe DNA Gel Stain (10 000X) in the hot melted agarose gel.
Let cool down for about 5 min.
Prepare the electrophoresis chamber to cast the gel, place the comb with the appropriate number of wells
Cast the gel inside the chamber and let cool for about 1 hour.
Samples preparation and loading on the agarose gel
Add the DNA 6X loading dye (4 μl for 24 μl of total volume) to the digested samples, mix with pipetting.
Add some TAE Buffer 1X on the gel to fill the wells before loading.
Load the sample on the gel
Fill the chamber with TAE 1X Buffer
Agarose gel electrophoresis
Assemble the electrophoresis unit. Make sure the anode cable (black) is plugged in the loaded DNA side (negative charges with negative charges for migration effect).
Run at 100 V for about 1h. Check at the image illuminator, run until the separation is good enough
Once the DNA separation is satisfying, remove the gel from the chamber and take a good image at the illuminator.
Prepare the scale and stirrer as well as the chemicals under the fume hood
Put 42.5mL of water in the 250mL beaker
Weigh 5g of NaOH and carefully add to the water under the fume hood
Weigh 2.5g of thiourea and carefully add to the NaOH solution under the hood
Weigh 5g of cellulose powder and add to the solution
Add the magnetic stirrer and stir at 750 rev min-1 to disperse cellulose homogeneously, making sure to check regularly. Wait until it’s homogenious
Preparation of cellulose hydrogel
Pour the resulting mixture into the mold
Freeze the solution at -20 degrees celsius for 24 hours
Thaw the mixture at room temperature
Rinse the obtained hydrogel with distilled water at least 3 times
Immerse the obtained hydrogel in ethanol
Leave the immersion at room temperature for 6 hours
Repeat steps 12 and 13 four times, meaning there are 5 solvent exchange steps in total
Critical Point Drying process
Dry via supercritical CO2 using a Tousimis Automegasamdri 915B critical point dryer. The chamber needs to be filled with ethanol before placing the gel inside. The machine needs to be set up to 40°C with a pressure of 8.96 MPa
Aerogel Proteins
Hydrogel coating
Materials
mSA-GFP-CBD (03a) protein purified
mSA-Silk-CBD (01a) protein purified (if needed in the experiment)
GFP (control) protein purified re-diluted in the wash Buffer A (300mM NaCl + 20mM Hepes) to have a same concentration as mSA-GFP-CBD (03a)
Double-distilled water
Already made hydrogels
Fluorescence microscope
Fluorescence Gel Imager
Protocol
Calculate the amount of protein needed to soak the volume of hydrogel you have.
Add the calculated amount of liquid on top of the aerogel.
Leave it for approximately 15 min and observe the result under the fluorescence microscope.
Take off the proteins and add new ones again as in step 2.
Take some images immediately after the soakings with the fluorescence microscope.
Leave for approximately 15 min and observe the result under the fluorescence microscope.
Place all three samples in a freezer for at least 8 hours (left overnight at a temperature of -20°C).
Take the samples towards the FD facility in an isotherm sagex container having ice in it.
Place the samples in a Christ Alpha lyophilizer at -50°C corresponding to a pressure 0.040 mbar for 24 hours.
Biotinylation
Materials
NHS-PEG4-Biotin, No-Weigh Format
PBS
Zeba Spin Desalting Column 5mL
HABA
Affinity purified Avidin 10mg
The protein we want to biotinylate
Protocol
Biotin Labeling Reaction
Cut off one microtube of NHS-PEG4-Biotin from the No-Weigh Microtube Strip. Return the unused strip of microtubes to its pouch and store desiccated at 4°C.
With a pipette tip, puncture the foil top on the biotin reagent microtube; add 170μL of water and mix by pipetting up and down to prepare a 20mM solution of NHS-PEG4-Biotin.
Add the appropriate volume of NHS-PEG4-Biotin solution to the protein solution.
Incubate reaction at room temperature for 45 minutes. There is no harm in reacting longer than the specified time other than the possibility of ordinary protein degradation or microbial growth.
Buffer Exchange and Remove Excess Biotin Reagent Using a Desalting Column
Prepare a Thermo ScientificTM ZebaTM Spin Desalting Column by breaking off the bottom plug and placing the column into a 15mL collection tube. Centrifuge the column at 1000 × g for 2 minutes, discard the storage buffer and return column to the same collection tube. Place a mark on the side of the column where the compacted resin is slanted upward. Place column in centrifuge with the mark facing outward in all subsequent centrifugation steps.
Equilibrate the column by adding 2.5mL of PBS to the top of the resin bed and centrifuging at 1000 × g for 2 minutes.
Discard the flow-through and repeat this step 2-3 times.
Place column into a new 15mL collection tube and apply protein sample directly onto the center of the resin bed. Allow sample to absorb into the resin.
Centrifuge the column at 1000 × g for 2 minutes. The collected flow-through solution is the purified protein sample.
HABA Assay for Measuring the Level of Biotin Incorporation
Pipette 180μL of HABA/Avidin Solution into a microplate well.
Measure the absorbance at 500nm of the solution in the well and record the value as A500 HABA/Avidin.
Add 20μL of biotinylated sample to the well containing the HABA/Avidin Solution. Mix the plate using an orbital shaker or plate mixer.
Measure the absorbance at 500nm of the solution in the well. Once the value remains fairly constant for at least 15 seconds, record the value as A500 HABA/Avidin/Biotin Sample.
Silk Biofilm
The following protocol is descripted in a general way in the dissertation of Felix Bauer12
Our recombinant silk protein 01a (mSA-N[AS]4C-CBD) purified
Protocol
Biofilm preparation
Dissolve 1% of (w/v) mSA-N[AS]4C-CBD (recombinant silk protein) in formic acid.
To determine the amount of protein required depending on the volume of formic acid (or vice versa), the online Percent Solutions Calculator from PhysiologyWeb can be used.
We have a concentration of 0.715 mg/ml for 01a silk protein. We have at our disposition 3.5 ml of 01a protein solution, which corresponds to m_silk = 0.715 mg/ml x 3.5 ml = 2.5025 mg. So if we want a 1% (w/v) solution of silk protein in formic acid, we need the following volume of formic acid:
V_formic_acid = 100 [μl/mg] x m_silk [mg] = 250.25 μl Reminder: the units of % (w/v) are in kg/L, or g/ml or mg/μl. If we want to use 2.5 ml of proteins to have 1 ml left, so 1.7875 mg, this corresponds to 178.75 μl of formic acid.
Cast formic acid on polystyrene (petri dish).
Drying step
Dry the film with an airing chamber for 24h (let the lid of the petri dish open).
Blaise Fleury, Eldho Abraham, Joshua A. De La Cruz, Varun S. Chandrasekar, Bohdan Senyuk, Qingkun Liu, Vladyslav Cherpak, Sungoh Park, Jan Bart ten Hove, and Ivan I. Smalyukh (2020)
Aerogel from Sustainably Grown Bacterial Cellulose Pellicles as a Thermally Insulative Film for Building Envelopes
