iBowu-China has compiled an account of everything that has happened in the lab over the summer. We are excited to show you how busy we have been in iGEM 2020! We believe that sharing our notes in labs will convey the passion and dedication of our team members, as we continuously push our limits to achieve high-quality results.
1. Preparation of Liquid LB Solution for E. Coli Competent Cell culture Petri Dishes
Preparation of Agarose Gel
Ingredients
100ml
250ml
NaCl
1g
2.5g
Tryptone
1g
2.5g
Yeast
0.5g
1.25g
Agarose
1.5g
3.75g
Kana Antibiotic
100μl
250μl
1.Solid LB solution prepared
2.Carefully pour the solution into clean petri dishes in clean benches
3.Allow the solution to dry over 30 minutes
4.Seal the petri dishes with tape
5.Store in 4 ̊C fridge, upside down to prevent accumulation of moisture
2. Gel Electrophoresis
Ingredients
1×
1.5×
LE Agarose
0.3g
0.45g
TAE
30ml
30ml
Yeast
0.5g
1.25g
M5 Gelred (nucleic acid dye)
2μl
2μl
1.Weigh the LE Agarose with an electronic balance.
2.Pour respective mass of agarose into the conical flask.
3.Pour respective volume of TAW into the conical flask.
4.Heat the solution with microwave for 1 minute.
5.If the agarose is completely dissolved, proceed to step 6. If the agarose is not completely dissolved, repeat step 4.
6.After cooling, add respective volume of nucleic acid dye to the solution.
7.Set up the gel mold.
8.Gently pour the gel inside the mold, making sure the liquid is evenly spread.
9.Insert the comb of the selected specification into the positioning slot of the glue making rack.
10.After the gel has solidified (30-60min), gently pull out the comb, take the gel out and put it in the electrophoresis tank.
Loading DNA samples
Use a pipette to slowly add the DNA sample vertically to the loading hole until below the opening.
Gel
1.Emerge the gel in the TAE buffer, making sure the liquid covers all the wells.
2.Make sure the wells are placed toward the cathode.
3.Pipette 20% volume of 6x Loading buffer into the samples, blend with vortex.
4.Always put a marker in the leftmost lane.
5.Add sample that is well mixed with loading buffer into the gel, record the order on the lab note.
6.Electrophoresis should last for 30min, 80V, 220microamps.
7.If Marker appears too short, run the gel for an additional 30 minutes with close monitoring.
8.After electrophoresis, obtain an image with the gel inspection machine and smart phone.
3. Plasmid Extraction (Omega E.Z.N.A. Plasmid DNA Mini Kid I)
1.Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1- 5 mL LB medium containing the appropriate selective antibiotic. Incubate for ~12-16 hours at 37°C with vigorous shaking (~ 300 rpm). Use a 10-20 mL culture tube or a flask with a volume of at least 4 times the volume of the culture. It is strongly recommended that an endA negative strain of E. coli be used for routine plasmid isolation. Examples of such strains include DH5a® and JM109®.
2.Centrifuge at 10,000 x g for 1 minute at room temperature
3.Decant or aspirate and discard the culture media
4.Add 250 μL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields
5.Transfer suspension into a new 1.5 mL microcentrifuge tube
6.Add 250 μL Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minutes incubation may be necessary.
7.Add 350 μL Solution III. Immediately invert several times until a flocculent white precipitate form.
8.Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step
9.Prepare the vacuum manifold according to manufacturer’s instructions
10.Connect the HiBind® DNA Mini Column to the vacuum manifold
11.Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind® DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind® DNA Mini Column
12.Turn on the vacuum source to draw the sample through the column
13.Turn off the vacuum
14.Add 500 μL HBC Buffer.
15.Turn on the vacuum source to draw the buffer through the column
16.Turn off the vacuum
17.Add 700 μL DNA Wash Buffer.
18.Turn on the vacuum source to draw the buffer through the column
19.Turn off the vacuum
20.Repeat Steps 17-19 for a second DNA Wash Buffer wash step
21.Transfer the HiBind® DNA Mini Column to a 2 mL Collection Tube
22.Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
23.Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube
24.Add 30-100 μL Elution Buffer or sterile deionized water directly to the center of the column membrane.
25.Let sit at room temperature for 1 minute.
26.Centrifuge at maximum speed for 1 minute.
27.Store DNA at -20°C
4. Gel Extraction
1.Carefully excise the DNA fragment of interest using scalpel from gel. Minimize the size of the gel slice by removing extra agarose.
2.Weighing the volume of the gel slice in a clean 1.5 mL microcentrifuge tube. Assuming a density of 1g/mL.
3.Add 1 volume XP2 Binding Buffer. (A gel slice of mass 0.3g will have a volume of 0.3mL)
4.Incubate at 50-60°C for 7 minutes or until the gel has completely melted. Vortex or shake the tube every 2-3 minutes.
5.Insert a HiBind® DNA Mini Column in a 2mL Collection Tube.
6.Add 700μL DNA/agarose solution from step 5 to the HiBind® DNA Mini Column.
7.Centrifuge at 10,000×g for 1 minute at room temperature.
8.Discard the filtrate and reuse collection tube.
9.Repeat Steps 6-8 until all of the sample had been transferred to the column.
10.Add 300μL XP2 Binding Buffer.
11.Centrifuge at maximum speed (≥13,000xg) for 1 minute at room temperature.
12.Discard the filtrate and reuse collection tube.
13.Add 700μL SPW Buffer.
14.Centrifuge at maximum speed for 1 minute at room temperature.
15.Discard the filtrate and reuse collection tube.
16.Repeat Steps 13-15 for a second SPW Buffer wash step.
17.Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
18.Transfer the HiBind® DNA Mini Column to a clean 1.5mL microcentrifuge tube.
19.Add 25μL Elution Buffer or deionized water directly to the center of the column membrane.
20.Set at room temperature for 2 minutes
21.Centrifuge at maximum speed for 1 minute.
22.Store DNA at -20℃.
5. Polymerase Chain Reaction
1.Set up a PCR system.
2.Place in PCR machine and set the program as needed.
3.30min before PCR ends, make agarose gel for DNA recycling.
4.Add 6x Loading Buffer into each system.
5.The first lane of the left-hand side should always be the Marker.
PCR System(50μL)
Ingredients
Volume
5*Prime Star Buffer
10 μl
dNTP
4μl
Forward Primer
1.5μl
Reverse Primer
1.5μl
DNA template
1μl
Prime Star Polymerase
1μl
ddH2O
31μl
PCR System(50μL)
Pre-Denaturing
98°C, 2 min
1 cycle
Denaturing
98°C, 15s
35 cycles
Annealing
55°C, 15s
35 cycles
Extending
68°C, 6min
35 cycles
Further Extending
68°C, 5 min
1 cycle
6.Gibson Assembly
1.Design plasmid and order primers..
2.Generate DNA segments by PCR.
3.Run PCR product on an agarose gel to check for size and yield. If there are significant amounts of undesired product, gel purify DNA segments.
4.Combine segments in Gibson Assembly Reaction.
Gibson Assembly System
Ingredients
20μL
10μL
2xGB mix
2μL
1μL
Linearized vector
2μL
1μL
PCR segment
2μL
1μL
water
6μL
3μL
*2-3 Fragment Assembly
5.Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts.
6.Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
7.Transform the DNA into bacteria and screen for the correct plasmid product by Restriction Digest.
7.Transformation of Plasmid DNA into Competent E. coli
1.Take 200μl of competent cell suspension from the -70℃ refrigerator and thaw it on ice for 1-2 min.
2.Add plasmid DNA solution (content not more than 50ng, volume not more than 10μl), shake gently, and place on ice for 30 minutes.
3.Heat shock in a 42℃ water bath for 90 seconds, and then quickly put it on ice for 2 minutes, without shaking the bacterial solution during the whole process.
4.Add 200μl LB liquid medium (without antibiotics) to the tube, mix well and culture with shaking at 37°C (225 rpm) for 1 hour to restore the normal growth of bacteria and express the antibiotic resistance gene encoded by the plasmid.
5.Shake the above bacterial liquid and spread it on an antibiotic-containing LB agar plate, and place it face up for half an hour. After the bacterial liquid is completely absorbed by the medium, invert the culture at 37°C for 12-16 hours.
8.HEK293T Cell Culture and Transfection
HEK293T cells are maintained in high-glucose DEME (Dulbecco’s Modified Eagle Medium, Thermo Fisher Scientific) with 1% P/S (Penicillin-Streptomycin, Thermo Fisher Scientific) and 10% FBS (fetal bovine serum, Thermo Fisher Scientific). Before seeding, the cells are cultured on flask. Then, cells are plated at 50,000 cells per well in a 6-well plate. After overnight incubation, cells are transfected by using Lipofectamine 2000 (Thermo Fisher Scientific).
9.Protein Crystals Purification
Pipetted out medium from wells, and rinsed once with sterile water. Added lysis buffer(1% NP-40 in 0.1M HEPES at pH 7.4)directly to the cell culture well and scraped cells off the surfaces. Pipetted the resulting cells into labeled eppendorf tubes and placed on a rocker (KS250 basic, Ika labortechnik Staufen)at room temperature for 40 minutes. After rocking, centrifuged at 2000 rpm for 1 minute on a benchtop centrifuge(Micro Star 12, VWR). Discarded supernatant and resuspended pellet in 0.1M HEPES buffer. Check crystal yield by pipetting a few microliters of suspension onto a coverslip and observing under the microscope.
10.Iron Loading
For iron loading, added fresh prepared FeSO4 solution (50 mM, 100X) to a 100 μL aliquot of the crystal suspension, incubated at room temperature for 2 hours. The final suspension changed color to pale yellow. To verify iron loading, freshly prepared the Prussian Blue (PB) stain (Sigma-Aldrich), pipetted a 10 µL droplet of the iron-loaded crystals onto a glass slide or coverslip and added 1 µL of the PB stain. The droplet can be immediately checked by brightfield optical microscopy.
11.MFM
Magnetic force microscopy (MFM) measurements were performed by a Bruker Dimension Icon (Santa Barbara, CA, USA). Before the MFM experiment, normal atomic force microscopy (AFM) experiment in tapping mode was carried out on the samples to reveal the accurate surface topography. The probe used for imaging the surface topography was a non-magnetic probe (Olympus). After the topographic measurement, lifted the cantilever up at a certain distance above the surface of the sample in the second pass to measure the magnetic force between the probe and the sample. Magnetic measurement was conducted using a magnetic MESP probe (Bruker) with the tip radius of ~ 35 nm, the normal spring constant of ~ 2.8 N/m, and the resonance frequency of ~ 75 kHz. Magnetic data was recorded by changes in the amplitude and phase of the probe oscillation.
