Team:Nanjing NFLS/Notebook
Notebook.
Go to "Project" and "Notebook" from bottom navigation bar.
Team Calendar
Lab Journal
2018.3.27-2018.4.14
We amplified ZsGreen fragment from plasmid and fused with artificially synthesized telomere and TK miniPRO fragments to generate plasmid T-TMEP and digested to generate sTMEP.
2018.4.14-2018.5.3
We tested the sTMEP together with synthesized TsgRNA and dCas9-VP64 on HepG2 and HL7702 cell lines and analyzed the experiments results.
2018.5.3-2018.5.23
We amplified Cas9 fragments, cloned into plasmid with artificially synthesized telomere and TK mini PRO fragments to generate T-TMCP and digested to be sTMCP.
We mutated wt CMV and did fusion PCR amplification with designed primers.
2018.5.23-2018.6.18
We tested the sTMCP together with synthesized TsgRNA and dCas9-VP64 on HepG2, HeLa, A549, Hep1-6, 293T and HL7702 cell lines and analyzed the experiments results.
We constructed pEGFP vector and cloned with wt CMV and mut CMV to transfect CHO and HepG2 cells.
2018.6.18-2018.7.13
We generated double-stranded DNA with 4-base overhang and complementary oligonucleotides with HO site to generate plasmid T-HOsite-sTMEP and T-HOsite-sTMCP.
We constructed the C1-HO plasmid with HO genes.
2018.7.13-2018.8.8
We tested the HOsite-sTMEP together with C1-HO and dCas9-VP64 on HepG2 and HL7702 cell lines and analyzed the results. We conducted Dual-Luciferase Reporter Assay and Gluc Reporter Assay to test the activity of mut CMV. We completed the experiments for InterLab study.
2018.8.8-2018.8.23
We amplified TsgRNA and cloned into plasmid pcDNA-dCas9-VP64 to generate TsgRNA-dCas9-VP64 in order to simply the system.
We redid the experiments for InterLab study.
2018.8.23-2018.9.15
We tested TsgRNA-dCas9-VP64, C1-HO, HOsite-sTMCP on HepG2 and HL7702 cell lines and analyzed the results.
We analyzed and made conclusion on all results we obtained from experiments.
Protocols
Plasmid Extraction Protocol
1.Pellet 1.5mL of overnight culture in a microfuge tube by centrifugation at 10,000 rpm for 2 minutes.
2. Discant the supertanant completely and add 200ul of resuspension buffer to the pellet.
3. Add 250uL of lysis buffer, invert the tube the tube 4-6 times to mix thoroughly until the solution becomes clear. The time should be no longer than 5 minutes.
4. Add 350uL of neutralization buffer, mix gently by inverting the tube 6-8 times or until a precipitate forms.
5. Centrifuge at 10,000rpm for 10 minutes.
6. Place spin column into a 2mL collection tube. Transfer supernatant in the step above to the column.
7. Centrifuge the spin column at 7,000rpm for 1 minute. Discard the flow through.
8. Return the column to the 2mL microfuge tube and add 400uL of wash buffer. Centrifuge at 7,000 rpm for 1 minute. Discard the flow through.
9. Return the column to the 2mL microfuge tube and add 400uL of wash buffer. Centrifuge at 7,000 rpm for 1 minute. Discard the flow through.
10. Centrifuge for an additional 2 minutes at 10,000 rpm to remove residual wash buffer.
11. Transfer the column into a clean 1.5mL microfuge tube. Add 60uL of eluent buffer to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 10,000rpm for 1 minute.
PCR Amplification of DNA fragments from plasmid Protocol
1.Add 73 ddH2O, 20uL S15buffer, 2 uL dNTPmix, 1 uL forward primer, 1 uL reverse primer, 1uL S15 DNA polymerase, 2 uLplasmid in order into a PCR tube.
2.Finger flick to mix, then centrifuge briefly.
3.Place the reaction tubes in the Thermal Cycler.
4.PCR will be run with the following programme:
1 cycle of 96 °C, 5 min
25cycles of 96 °C, 30 sec; 58 °C, 30 sec; 72 °C, 30-50 sec (depend on the size of DNA fragment)
Final extension 72 °C, 2 min
Hold at 4 °C
5.Check PCR results on an Agarose gel
Agarose Gel Electrophoresis Protocol
Gel preparation:
1.Prepare sufficient electrophoresis buffer (1:10 dilution of TBE:distilled water)
2.Clean a plastic tray. Position the comb 0.5-1 mm above the plate so that a complete well is formed when the agarose is added.
3.Prepare agarose gel.
For a 2% agarose gel: measure 2 g agarose in an Erlenmeyer flask and add 100 mL1x TBE buffer.
4.Scale the flask and note its weight on it.
5.Cover the flask with kimwipes/ parafilm and heat with microwave until the agarose dissolves. Measure it again and complete the evaporated liquid with distilled water.
6.Stain the agarose solution: 5μL ECO Safe Nucleic Acid Staining Solution / 100 mL gel
7.Mix the agarose solution well by swirling the flask. Pour the agarose into the mold. (3-5 mm thickness)
8.Leave it to cool down to about 60 °C on the bench for several minutes
9. Carefully remove the comb after 30 minutes at room temperature.
10.Position the gel into the gel electrophoresis tank.
11. Add enough TBE buffer to cover the gel to a depth of about 5 mm.
Loading:
1. Mix the DNA samples with gel-loading buffer with pipettes: 5 μL of buffer + DNA solution (Note: about 0.3 - 0.5 μg of DNA gives a visible band or 1/10 of PCR reaction volume)
2. Prepare marker DNA of known size: 6 μl from Promega Marker
3.Load the mixtures slowly into the slots. (Avoid making bubbles!)
4.Attach the electrical leads so that DNA can move toward the anode (red lead).
Apply a voltage of 1-5V/cm.
5.Run the gel until the gel-loading buffer stain has migrated the appropriate distance (normally until the bromophenol blue dye front migrated 3⁄4 of the way down the gel).
DNA Gel Extraction Protocol
1.Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5mL tube and weigh. Record the weight of the gel slice.
2.Put EB (elution buffer) at 65 degree water bathing.
3.Add a 3:1 volume of Solution Buffer to the gel slice (volume : weight) (e.g., add 300uL of Binding Buffer for every 100 mg of agarose gel). Incubate the gel mixture at 60 degree for 5 min at least until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. 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 10uL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
4.Pour the solution to a fresh adsorption column. Centrifuge at 13,000 rpm for 1min. Pour off the liquid in the collection tube. For critical samples, repeat the operation above.
5.Add 600uL washing buffer before centrifugation at 13,000 rpm for 1 min. Pour off the liquid into beaker.
6.Centrifuge at 13,000rpm for 10 min to spin the ethanol down.
7.Put the column into a fresh EP tube. Air-dry the pellet for 10-15min to avoid the presence residual ethanol in the purified DNA solution if necessary. Residual of ethanol in the DNA sample may inhibit downstream enzymatic reactions.
8.Add 30-50uL elution buffer to elute the DNA.
9.Get 5uL of the eluted sample to identify with electrophoresis.
Restriction Enzyme Digestion and DNA Ligation Protocol
1. Enzyme Digestion
To a 1.5mL microfuge tube, add
2.DNA ligation
To a 1.5mL microfuge tube, add
DH5alpha Competent Cells Preparation Protocol
1. Inoculate 5mL LB broth with an aliquot of the desired E.coli from the -80°C freezer stock of cells in 50mL falcon tube.
2. Incubate for 2h at 37°C.
3. Add the 2mL seed culture to 250mL LB broth and grow at 37°C, shake (about 200rpm) for 4-6 hrs.
4. Pre-cool CaCl2-glycerine (0.1mol/L CaCl2) and CaCl2- MgCl2 (80mmol/L MgCl2, 20mmol/L CaCl2). Set the centrifuge and prepare the ice tray.
5. Transfer the bacteria into the 50mL polypropylene tube. Place it on ice for 10 minutes.
6. Centrifuge at 4°C, 6,000rpm for 5 minutes.
7. Descant supernatant, add 20mL of pre-cooled CaCl2- MgCl2 per 50mL of initial liquid medium and gently resuspend bacteria cell pellet.
8. Centrifuge at 4°C, 6,000rpm for 5 minutes.
9. Discard supernatant, add 2mL of pre-cooled CaCl2 per 50mL of initial liquid medium and gently resuspend bacteria cell pellet.
10. Dispense in microtubes (50uL/tube). Freeze in -80°C.
Heat Shock Transformation Protocol
1.Take competent cells out of -80°C and thaw on ice for approximately 20-30min.
2.Mix 5μl of DNA into 50μL of competent cells in a microcentrifuge or falcon tube. Gently mix by flicking the bottom of the tube with finger a few times.
3.Incubate the competent cell/DNA mixture on ice for 20-30min.
4.Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 45 seconds.
5.Put the tubes back on ice for 2 min.
6.Add 500μl LB media and grow in 37°C shaking incubator for 60 min.
7. Activate on the plate for 60 minutes.
8. Centrifuge at 3,000rpm for 1 minute.
9. Operate in the clean bench, discard the supertanant and resuspend bacteria cells.
10. Use the inoculating loop to load bacteria liquid then streak on the LB plate.
v
11. Place plates upside down and incubate at 37°C overnight.
Cell Culture and Transfection Protocol
1. Maintain the cells in a humidified incubator set at 37°C and 5% CO2.
2. Culture Hepa1-6, HEK-293T, HepG2, HeLa cells in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone), supplemented with 10% fetal calf serum (FBS) (HyClone), 100 U/mL penicillin and 100 μg/mL streptomycin.
3. Culture A549, HL7702 in Roswell Park Memorial Institute (RPMI) 1640 Medium (HyClone), supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin.
4. Place DNA equally into individual tubes before transfection.
5. Seed cells into 24-well plates at a density of 0.5×105 cells/wells and transfect with DNA.
For each transfection:
1. Culture cells with 500 L of Opti-MEM (ThermoFisher Scientific) at 37 °C for 30 min when grown to a density of 2×105 cells/well.
2. Make a stock solution of 50 L of Opti-MEM, 500 ng of total DNA and 50 L of Opti-MEM, 2 L of Lipofectamine 2000 (ThermoFisher Scientific) per transfection respectively.
3. Vortex and incubate the solution respectively for 5 minutes at room temperature.
4. Add the Opti-MEM/Lipofectamine solution to the individual aliquots of DNA stocked in 50 L of Opti-MEM. Vortex, and incubate for 20 minutes at room temperature before being added to each well.
5. Incubate for 5 h, and replace the medium of each well with 500 L of fresh DMEM or RPMI 1640 medium containing 10% FBS.
6. Incubate the cells at 37 °C and 5% CO2 for another 24 h.
Cell Staining, Observation, Quantification Protocol
1. Stain the cells with Acridine organge.
2. Wash the cells with PBS, and stain the cells with Acridine orange (Solarbio) for 10 minutes at room temerapture.
3. Observe the photograph the cells with a fluorescent microscope (Olympus) at the constant magnification of 200×.
4. Quantify the fluorescence intensity of cells with a flow cytometry (Calibur, BD, USA).
5. Calculate the mean fluorescent intensity (MFI) by BD software of flow cytometry.