Team:CSU CHINA/Experiments
Design
Thaw 50µL DH10B competent E. coli cells on ice.
Add 5 µl DNA from a ligation reaction mix or 10-100ng DNA of a plasmid.
Place the mixture on ice for 20-30 minutes.
Heat shock at 42°C for 90 seconds.
Place on ice for 5 minutes.
Pipette 200 µl of room temperature LB media into the mixture.
Incubate at 37°C and 220 rpm for 60 minutes.
Add 50-100 µl of the transformed cells to the selection plate.
Overnight cultures were prepared under sterile conditions using a Bunsen burner
Add 3 mL liquid LB media into test tubes
Add 3 μL of appropriate antibiotic（1000x） into the broth
Using the pipette tips, pick a single colony and inoculate the cultures by dipping the tip into the LB broth or by adding 50 μL stored cells
Put caps on the tubes and incubate overnight at 37°C shaking at 200-250 rpm
In a PCR tube on ice, combine 1-10 ng of plasmid DNA or just 1 µL template, 2 µL of 10 µM forward primer, 2 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Phanta Mastermix, and sterile water up to 50 µL.
Gently mix the reaction
If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
Transfer the PCR tube from ice to a PCR machine preheated to 98°C to begin thermocycling
Add 10 g Tryptone, 5 g Yeast Extract and 10 g Sodium Chloride to 1 litre purified water
Autoclave
Add 10 g Tryptone, 5 g Yeast Extract, 10 g Sodium Chloride and 20 g Agar to 1 litre purified water
Autoclave
Add vector plasmid, insert DNA, T4 DNA ligase and T4 DNA ligase buffer to the microcentrifuge tube
Make reaction up to 10 µL using sterile water
Incubate at room temperature for 30 - 60 min
Set up the reaction following the instruction below, depending on whether test digest or assembly digest is being performed.
For a Test digest (10 µL):
5 µL Plasmid DNA
0.5 µL Restriction Enzyme 1
0.5 µL Restriction Enzyme 2
1 µL 10× buffer
Add sterile water to 10 µL
For gene assembly(30 µL):
15 µL Plasmid DNA
1.2 µL Restriction Enzyme 1
1.2 µL Restriction Enzyme 2
3 µL 10X buffer
Add sterile water to 30 µL
Incubate digestion reaction at 37°C for 30 minutes-1 hour
Perform heat deactivation at 80°C for 20 minutes, if not running on a gel at the end of incubation.
1.Keep 15µL Gibson mix on ice.
2.Add 5µL DNA fragment (Vector fragment: less than 15ng; small fragment: 20-50ng).
3. Incubate at 50℃ to 60min.
Store in -20℃, aliquot 15µL
Add ddH2O to 6mL, store in -20℃,Aliquot 100µL
Prepare 1% w/v solution of agarose powder in 1×TAE buffer (e.g. 1g agarose powder in 100 mL buffer) using a conical flask.
Heat the mixture until agarose is completely dissolved.
Add 5 µL EB to the solution.
Pour the solution into a gel mould.
Allows the solution to set (approx 15-20 minutes)
Transfer the agarose gel to a tank, remove the comb and apply:
3-5 µL of the DNA ladder
DNA samples with the corresponding amount of DNA loading buffer (6X)
Run the gel for 20 minutes at 120V.
PCR Purification, Gel Extraction & Miniprep
PCR purification was performed according to the Generay PCR Purification Kit
Gel extraction was performed according to the Generay Gel Extraction Kit
Plasmid extraction were carried out according to the Magen Miniprep Plasmid Kit
1.Prepare 3mL bacteria solution grown overnight in LB broth, centrifuge at 10,000×g for 1min. Discard the supernatant.
2.Add 250µL Buffer P1/RNase A to resuspend bacteria cells and transfer to a 1.5mL centrifuge tube.
3.Add 250µL Buffer P2 and gently invert the tube 8-10 times to mix.
4.Add 350µL Buffer P3 and gently invert the tube immediately but gently 8-10 times.
5.Centrifuge for 10min at 13,000×g in a microcentrifuge.
6.Apply the supernatants from step 5 to the HiPure DNA Mini Column Ⅱspin column by pipetting.
7.Centrifuge for 1min at 13,000×g. Discard the flow-through.
8.Wash the HiPure DNA Mini Column Ⅱspin column by adding 500µL Buffer PW1, and centrifuging for 1min at 13,000×g. Discard the flow-through.
9.Wash the HiPure DNA Mini Column Ⅱ by adding 600µL Buffer PW2 (diluted with absolute ethanol), and centrifuging for 1min at 13,000×g. Discard the flow-through.
10.Repeat the step 9.
11.Centrifuging for an additional 1min at 13,000×g to remove residual wash buffer.
12.Place the HiPure DNA Mini Column Ⅱ in a clean 1.5mL microcentrifuge tube. Add 50µL Elution Buffer (65°C) to the center of the HiPure DNA Mini Column Ⅱ, let stand for 1min, and centrifuge for 1min at 13,000×g. For better production, repeat Step12 for a second time.
13.Discard the column，measure the concentration of plasmid by Nanodrop2000，then preserve it at -20°C.
1.Remove culture medium from the dish. Gently add 1 ml of phosphate buffered saline (PBS) to the dish, and shake it several times.
2.Remove PBS from the dish. Add 1 ml of pre-warmed 0.1% Trypsin to the dish. Gently shake it to get complete coverage of the cell layer. Incubate the cells at room temperature for 40-60 seconds. (Note: The actual incubation time varies with the cell line used.)
3.Add 2ml of pre-warmed complete growth medium (DMEM with 10% FBS). Disperse the cells by pipetting over the cell layer surface several times.
4.Transfer the cells to 15 ml centrifuge tubes and centrifuge at 1200 ×g for 3 minutes.
5.Resuspend the cell pellet in 1-2 ml of pre-warmed medium and take 10 μl of medium for cell counting.
6.Cells were seeded in plates at the recommended density, and pipe the rest of the cells into new cell culture dishes, and put dishes back to the incubator (37°C, 5% CO2).
1. Thaw FBS. Make sufficient 10% FBS in Opti-MEM media (w/o antibiotics) sterile filter
2. Aspirate medium off cells
3. Add 2-3 ml PBS to wash
4. Aspirate off PBS
5. Add 8 ml of 10% FBS/ Opti-MEM media /plate
6. Incubate 30min at 37°C while mixing following components in hood (see spreadsheet
for amounts, this example is for one 10cm plate):
a. A Tubes: Add selected DNA, and Q.S. w/ Opti-MEM to 100µl
b. B Tubes: Add LF2000 and Q.S. with Opti-MEM to 100µl
c. Incubate in hood for 5 min
7. Add Component B to A by gently bubble mixing
8. Incubate A/B complexes at RT for 30 min
9. Drip 200µl A/B complex mixture onto cells respectively
10. Gently mix media on cells via figure 8 pattern and back and forth motions
11. Incubated 37° for 48 hours
12. Remove Opti-MEM media containing A/B complexes and discard
13. Add fresh 8ml/ plate of 10% FBS/DMEM with antibiotics
1. Remove growth medium from cultured cells.
2. Rinse cells in 1X PBS. Do not dislodge cells. Remove as much of the final wash as possible.
3. Dispense a minimal volume of 1X lysis reagent (CCLR, RLB or PLB) into each culture vessel (e.g.,400µl/60mm culture dish, 900µl/100mm culture dish or 20µl/well for a 96 well plate).
4. For culture dishes, scrape attached cells from the dish, and transfer the cells and solution to a microcentrifuge tube. Pellet debris by brief centrifugation, and transfer the supernatant to a new tube.
5. Mix 20µl of cell lysate with 100µl of Luciferase Assay Reagent and measure the light produced.
Single-tube Luciferase reporter assay
HepG2, LO2, Huh7 and MCF7 cells were cultured in DMEM (GIBCO) supplemented with 10% FBS. All cells were cultured at 37°C and 5% CO2. For transfection, HepG2, LO2, Huh7 and MCF7 cells were seeded at 4 × cells/well in 24‐well plates and transiently transfected by the lipo2000-mediated method. Briefly, each well is transfected with 15-30ng of constructed plasmid on two consecutive days.
Luciferase assays were performed as described previously. Briefly, the cells were transfected with 100 ng of the luciferase (firefly) reporter plasmid as designed containing different promoter into 24-well plate. After 48h transfection, cells were collected and lysed. Firefly luciferase activities were measured with the single-tube luciferase Reporter mechine and β‐galactosidase luciferase activity was used to normalize for potential differences in transfection efficiency.
We use a series of methods above and fusion enzyme to construct targeted and mutated plasmid.
Results are expressed as mean ± SE. Differences between two groups were assessed using multiple two‐tailed Student's t‐tests（*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001）. Statistical calculations were performed using Graphpad Prism 6.0.
*For experiment development, you can turn to notebook to get the detail information
