Difference between revisions of "Team:TJU China/Experiments"

1.Substrate Preparation
In the ventilation table, remove the substrate, put into a one-off cell culture dish (10cm).
If necessary, use water and ethanol circulation ultrasonic to wash glass, high pressure sterilization, and dry. Then, wrap them with tin foil in the box.
2.Place hydrophobic protein
  ①Use a special pen to wrap the liquid to prevent dispersing into too large circles. Each circle is dripped with 1-5ul hydrophobic protein
  ②Dry in a ventilated place dry, and use depc water to slowly rinse。

4. Mix NEB buffer 3, crRNA, FnCas12a protein and Nuclease-Free Water thoroughly and pulse-spin in a microfuge, pre-incubate at 25⁰C for 15 minutes.
5. Incubate for 1 hour at 37⁰C.
6. Proceed with fluorescence microscope detection.
Notice: X, Y, Z, W, M in the table above are various according to our experiment design.

1. Prepare sufficient lx TAE to fill the electrophoresis tank and to cast the gel.
2. Prepare a solution of agarose in electrophoresis buffer at a concentration of 1%: Add 0.9g powdered agarose to 90ml of TAE in an Erlenmeyer flask. （for the gel electrophoresis of the crRNA，the final concentration of the agarose is 5%.）
3. Heat the slurry in a boiling-water bath or a microwave oven until the agarose dissolves.
4. Use insulated gloves or tongs to transfer the flask/bottle into a water bath at 55°C. When the molten gel has cooled, add ethidium bromide to a final concentration of 0.5 ug/ml. Mix the gel solution thoroughly by gentle swirling.
5. While the agarose solution is cooling, choose an appropriate comb for forming the sample slots in the gel. Position the comb 0.5-1.0 mm above the plate so that a complete well is formed when the agarose is added to the mold.
6. Pour the warm agarose solution into the mold.
7. Allow the gel to set completely (30-45 minutes at room temperature), then carefully remove the comb. Pour off the electrophoresis buffer and carefully remove the tape Mount the gel in the electrophoresis tank.
8. Add just enough electrophoresis buffer to cover the gel to a depth of ~1 mm.
9. Mix the samples of DNA with 10 ul green buffer
10. Slowly load the sample mixture into the slots of the submerged gel using a disposable micropipette, an automatic micro-pipettor. Load size standards into slots on both the right and left sides of the gel.
11. Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate toward the positive anode (red lead). Apply a voltage of 1-5 V/cm (measured as the distance between the positive and negative electrodes). If the leads have been attached correctly, bubbles should be generated at the anode and cathode (due to electrolysis), and within a few minutes, the bromophenol blue should migrate from the wells into the body of the gel. Run the gel until the bromophenol blue and xylene cyanol FF have migrated an appropriate distance through the gel.
12. When the DNA samples or dyes have migrated a sufficient distance through the gel, turn off the electric current and remove the leads and lid from the gel tank.

1. Collect the E. coli solution into the EP tube.
2. Re-suspend pelleted bacterial cells in 250ul Buffer P1 (RNase A added, kept at 4 °C) and mix thoroughly.
3. Add Buffer 250ul P2 and gently invert the tube 6-8 times to mix.
4. Add 350μl Buffer P3 and invert the tube immediately and gently 6-8 times.
5. Centrifuge for 10 min at 12,000 rpm in a micro-centrifuge.
6. Regenerate column CP3 while centrifugation. Add 500μl Buffer BL. Centrifuge for 1 min at 12,000 rpm after static for 2min. Discard the flow-through.
7. Add supernatant from the EP tube to the column and put it into collection canals. Centrifuge for 1min at 12000rpm. Discard the flow-through.
8. Add 600μl Buffer PW(ethanol added) and centrifuge for 1min after static for 2min. Discard the flow-through.
9. Repeat step 8.
10. Centrifuging for 2min at 12000rpm to shake off the rest of the Buffer PW.
11. Place the column in a new EP tube and the opening was allowed to stand for 5 minutes, so that the ethanol in the PW can be sufficiently volatilized.
12. Add 70 ul sterile distilled water at 75℃ dropwise to the middle of the adsorbed film. Static for 2min. Then centrifuge for 2 min at 12,000 rpm to collect DNA solution in EP tube.

E. coli Rosetta competent cells were transformed with pET-Cas9-NLS-6xHis which was purchased from Addgene. The plasmid encodes the S. pyogenes Cas9 fused to a C-terminal His-tag and an N-terminal NLS. The resulting single colony was grown in Luria-Bertani (LB) media at 37 °C overnight, and the overnight culture (5 mL) was inoculated into LB (1 L) in the presence of ampicillin (100μg/ml) and chloramphenicol(100μg/ml) at 37℃ for 6h. The Cas9 protein was induced with 1 mM of isopropyl β-D-1-thiogalactopyranoside at 18℃ for 16 h. The cells were collected by centrifugation at 4,000rpm for 15 min at 4 ℃. The pellets were harvested, and resuspended in Buffer A(50 mM Tris(hydroxymethyl)aminomethane hydrochloride) (Tris-HCl, pH 8.0), 1 M NaCl, 20% glycerol, 20 mM imidazole and 2 mM Tris(2-carboxyethyl)phosphine (TCEP). The cells were lysed and the soluble lysate was obtained by centrifugation at 18,000r for 40 min at 4℃. The cell lysate was incubated with His-Pure nickel-nitriloacetic acid (nickel-NTA) resin at 4℃ for 30 min to capture His-tagged Cas9. The resin was transferred to a 20-ml column and washed with 20 column volumes of Buffer A. Cas9 was eluted in Buffer B( 50 mM Tris-HCl (pH 8), 1 M NaCl, 20% glycerol, 2 mM TCEP and 500 mM imidazole), and concentrated by Amicon ultracentrifugal filter (30-kDa molecular weight cut-off) to 1ml. The eluted Cas9 was loaded onto a HiTrap SP HP, 5ml and purified using a linear gradient of NaCl from 0.1M to 1M in buffer C (50mM Tris-HCl, pH8.0, 20% glycerol, 20 mM imidazole and 2mM TCEP). The collected liquid was concentrated to 1ml. The eluent, containing Cas9, was injected into a molecular sieve column，concentrated and exchange the buffer to Storage Buffer(300 mM NaCl, 10 mM Tris-HCl, 0.1 mM EDTA ,1 mM DTT ,50% Glycerol ,pH 7.4 at 25℃). Finally, the Cas9 protein was quantified by Bicinchoninic acid (BCA) assay, snap-frozen in liquid nitrogen and stored in aliquots at −80 °C.

1.Add a 1:1 volume of Binding Buffer to completed PCR mixture (e.g. for every 100 µL of reaction mixture, add 100 µL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
2.for DNA ≤500 bp
Optional: if the DNA fragment is ≤500 bp, add a 1:2 volume of 100% isopropanol (e.g., 100 µL of isopropanol should be added to 100 µL of PCR mixture combined with 100 µL of Binding Buffer). Mix thoroughly.
3.Transfer up to 800 µL of the solution from step 1 (or optional step 2) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through. Notes. If the total volume exceeds 800 µL, the solution can be added to the column in stages. After the addition of 800 µL of solution, centrifuge the column for 30-60 s and discard flowthrough. Repeat until the entire solution has been added to the column membrane. Close the bag with GeneJET Purification Columns tightly after each use!
4.Add 700 µL of Wash Buffer (diluted with the ethanol as described on p. 3) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through and place the purification column back into the collection tube.
5.Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.
6.Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included). Add 50 µL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min.
7.Discard the GeneJET purification column and store the purified DNA at -20 °C.

2.Mix thoroughly and incubate at 37℃ for 16h.
3.Proceed with purification.

1. Bring the RNA sample to 100 µL with Elution Solution. Mix gently but thoroughly.
2. Add 350 µL of Binding Solution Concentrate to the sample. Mix gently by pipetting.
3. Add 250 µL of 100% ethanol to the sample. Mix gently by pipetting.
4. Apply the sample to the filter
  a. Insert a Filter Cartridge into 1 of the Collection and Elution Tubes supplied.
  b. Pipet the RNA mixture onto the Filter Cartridge.
  c. Centrifuge for ~15 sec to 1 min, or until the mixture has passed through the filter. Centrifuge at RCF 10,000–15,000 × g (typically 10,000–14,000 rpm). Spinning harder than this may damage the filters.
  d. Discard the flow-through and reuse the Collection and Elution Tube for the washing steps.
5. Wash with 2 × 500 µL Wash Solution(ethanol added).
  a. Apply 500 µL Wash Solution. Draw the Wash Solution through the filter as in the previous step.
  b. Repeat with a second 500 µL aliquot of Wash Solution.
  c. After discarding the Wash Solution, continue centrifugation or leave the Filter Cartridge on the vacuum manifold for 10–30 sec to remove the last traces of Wash Solution.
6. Elute RNA from the filter with 50 µL Elution Solution.
  a. Pre-heat 110 µL of Elution Solution per sample to 95° C.
  b. Apply 50 µL of the pre-heated Elution Solution to the center of the Filter Cartridge, close the cap of the tube and centrifuge for 1 min at room temperature (RCF 10,000–15,000 x g) to elute the RNA.
  c. To maximize RNA recovery, repeat this elution procedure with a second preheated 50 µL aliquot of Elution Solution. Collect the eluate into the same Collection/Elution Tube.

1. Prepare sufficient lx TAE to fill the electrophoresis tank and to cast the gel.
2. Prepare a solution of agarose in electrophoresis buffer at a concentration of 1%: Add 0.9g powdered agarose to 90ml of TAE in an Erlenmeyer flask.
3. Heat the slurry in a boiling-water bath or a microwave oven until the agarose dissolves.
4. Use insulated gloves or tongs to transfer the flask/bottle into a water bath at 55°C. When the molten gel has cooled, add ethidium bromide to a final concentration of 0.5 ug/ml. Mix the gel solution thoroughly by gentle swirling.
5. While the agarose solution is cooling, choose an appropriate comb for forming the sample slots in the gel. Position the comb 0.5-1.0 mm above the plate so that a complete well is formed when the agarose is added to the mold.
6. Pour the warm agarose solution into the mold.
7. Allow the gel to set completely (30-45 minutes at room temperature), then carefully remove the comb. Pour off the electrophoresis buffer and carefully remove the tape Mount the gel in the electrophoresis tank.
8. Add just enough electrophoresis buffer to cover the gel to a depth of ~1 mm.
9. Mix the samples of DNA with 10 ul green buffer
10. Slowly load the sample mixture into the slots of the submerged gel using a disposable micropipette, an automatic micro-pipettor. Load size standards into slots on both the right and left sides of the gel.
11. Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate toward the positive anode (red lead). Apply a voltage of 1-5 V/cm (measured as the distance between the positive and negative electrodes). If the leads have been attached correctly, bubbles should be generated at the anode and cathode (due to electrolysis), and within a few minutes, the bromophenol blue should migrate from the wells into the body of the gel. Run the gel until the bromophenol blue and xylene cyanol FF have migrated an appropriate distance through the gel.
12. When the DNA samples or dyes have migrated a sufficient distance through the gel, turn off the electric current and remove the leads and lid from the gel tank.

The COS7-EGFP cell line, a generous gift from School of Public Health, University of South China, was used as the model cell line which has a single copy of destabilized EGFP gene integrated into the genome. The cells were cultured in a 37 °C incubator under 5% CO2 and 90% humidity with full serum medium: DMEM supplemented with 10% (v/v) FBS. COS7-EGFP cells were seeded into glass bottom cell culture dish (~60,000 cells per well) one day before the transfection. When the cells reached 70% confluence, the medium was replaced with fresh DMEM medium containing the BODIPY/sgRNA/Cas9 (or Liposome/sgRNA/Cas9) complex (various according to the experiment design below). After incubation for 4h, the Cas9 containing medium was replaced with fresh full serum medium incubated for another two days for analyzed by laser scanning confocal microscope (LSCM).

Vector:
Take 1.5 ng of the verified plasmid, add 1 μL Xba1 , 1μL Nhe1 ,5 μL 10X Fast Digest buffer , add ddHO to 50 μL, place in a 37 ° C water bath for 15min, then put in a 80 ° C water bath for 20 min.
Add 1μL Antarctic Phosphatase , 5.6μL Antarctic Phosphatase Buffer , then place in a 37 ° C water bath for 30min, then put in a 70 ° C water bath for 10 min.
Take out and run the gel verification.
Insert:
Take 1 ng of the verified insert fragment, add1 μL Xba1, 1μL Nhe1 ,5 μL 10X Fast Digest buffer , add ddHO to 50 μL, place in a 37 ° C water bath for 1h, then put in a 80 ° C water bath for 20 min, take out and run the gel verification.
connection:
COX8A: First add vector 100ng, insert 75ng, then add 1μL T4 ligase, 4 μL of its buffer, add ddHO to 20 μL, place in 22 ° C in a thermos bucket, and place in 4℃ connect overnight.
SOD2: First add vector 100ng, insert 75ng, then add 1μL T4 ligase , 4 μL of its buffer, add ddHO to 20 μL, place in 22 ° C in a thermos bucket, and place in 4℃ connect overnight.
ATP5: First add vector 100ng, insert 81.25ng, then add 1μL T4 ligase , 4 μL of its buffer, add ddHO to 20 μL, place in 22 ° C in a thermos bucket, and place in 4℃ connect overnight.
Conversion:
1. Wipe the cleaned work area with 70% ethanol.
2. Thaw the competent cells on ice. A 1.5 mL microcentrifuge tube was labeled for each transformation and the tube was placed on ice for pre-cooling. .
3. Inhale connection system into each microcentrifuge tube.
4. Pipette 50 μL of competent cells into each tube. Gently mix the tube with your fingers.
5. Incubate on ice for 30 minutes.
6. Heat the cells by placing in a 42℃ water bath for 90 seconds.
7. Immediately transfer the tube back to the ice and incubate on ice for 2 minutes.
8. Add 800 μL of LB medium to each tube, incubate at 37 ° C for 1 hour, and shake at 200-300 rpm.
9. Pipette bacteria from each tube onto the appropriate plate and spread the mixture
evenly over the plate. Incubate overnight or for about 16 hours at 37 ° C. Place the plate with the agar side on top and the lid on the bottom.