Team:Nanjing NFLS/Notebook

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• Project
  • Description
  • Background
  • Design
  • Experiment
  • Demonstrate
  • Notebook
  • Vision
• Parts
• Safety
• Model
• Team
  • Team Members
  • Attributions
  • Collaborations
• Human Practices
  • Overview
  • Integrated Human Practices
  • Public Engagement
• Judging

Notebook

• Notebook
• General protocols

Notebook

Date	Introduction
28-May-2019	Obtained   Plasmid extraction
02-Jun-2019	Preparation   of DH5α competent cells by CaCl2 method
03-Jun-2019	1.   Obtained pcDNA3.1, pcDNA6.2-HBsAg plasmid 2. Double   digestion of the pcDNA6.2-HBsAg plasmid and the hTERT plasmid for ligation
04-Jun-2019	Transferred   pcDNA6.2-HBsAg and hTERT linker into DH5α
15-Jul-2019	Performed   double digestion on the pcDNA3.1 plasmid and the Ce-125-GAAA plasmid for   ligation.
16-Jun-2019	1.   Transferred pcDNA3.1 and Ce-125-GAAA linker into DH5α 2.   Obtained the pcDNA6.2-hTERT-HBsAg plasmid
18-Jul-2019	Obtained   the pcDNA3.1-Ce-125-GAAA plasmid
23-Jul-2019	1.   Verified that pcDNA3.1-Ce-125-GAAA is successfully connected 2. Initial   verification of the correctness of the pcDNA6.2-hTERT plasmid 3.   Prepared DH5α competent cells (transformation) by CaCl2 method
30-Jul-2019	Double-cleared   the pcDNA6.2-hTERT-HbsAg plasmid and the miR-70 and miR-95 plasmids for   ligation
13-Aug-2019	Transferred   pcDNA6.2-hTERT-HBsAg to miR-70, miR95 linker into DH5α
15-Aug-2019	Double-digested   the pcDNA6.2-hTERT-HbsAg plasmid and the miR-125 plasmid for ligation
16-Aug-2019	Prepared   DH5α competent cells (transformation) by CaCl2 method
18-Aug-2019	Double-clearing   the pcDNA3.1 plasmid and the Ce-70-GACT and Ce-95-tctc plasmids for ligation
19-Aug-2019	Transferred   pcDNA3.1 and Ce-70-gact, Ce-95-tctc linker into DH5α
21-Aug-2019	1.   Obtained pcDNA3.1-Ce-70-gact, Ce-95-tctc plasmid 2.   Verified that pcDNA3.1-Ce-70-gact and Ce-95-tctc are successfully connected
27-Sep-2019	1.   Resuscitated 293T cells 2. Resuscitated   HepG2 cells
30-Sep-2019	1. Passed   the passage of the overgrown HepG2 cells. 2. Passed   the passage of the overgrown 293T cells.
02-Oct-2019	1.Passed the passage of the overgrown   HepG2 cells into the large bottle. 2. Passed   the passage of the overgrown HepG2 cells.
05-Oct-2019	1.Transfected PGL3-Basic, PGL3-Hulc,   PGL3-hTERT, and PGL3-Control plasmids. 2.   Transfected the PGL3 series plasmids
07-Oct-2019	Detection   of PGL3-Basic, PGL3-Hulc, PGL3-hTERT, PGL3-Control transfection plasmids

 

General protocols

Cell cryopreservation

1. Cryopreservation occurs when the cell density is as long as the cell bottle exceeds 80%.

2. The supernatant was discarded, and 1 mL of trypsin was added to gently shake the trypsin to infiltrate the cell bottle and discard it, and placed at 37 ° C for 1 min.

3. Under the microscope, when the cell morphology became round, it showed complete digestion.

4. Add 1 mL of medium to stop digestion (this step should be done quickly to avoid overdigestion).

5. The cells in the flask were aspirated into a 15 ml centrifuge tube at 1000 rpm for 5 min.

6. Discard the supernatant and add 6 mL of cryopreservation solution (90% complete medium + 10% DMSO)

7. Equally divided into 6 frozen tubes.

8. Quickly place in a freezer box and store at -80 °C.

 

Plasmid extraction

1. Add 250 μl of Buffer P1 containing RNase A to the cell pellet, mix well, and suspend the cell pellet.

2. Add 250 μl Buffer P2, gently mix upside down 4-6 times, mix well, this step can not exceed 5 minutes.

3. Add 350 μl Buffer N3, gently mix upside down 8-10 times, and mix well.

4. Centrifuge at 12,000 rpm for 10 minutes (precipitation was incomplete and centrifuged for another 10 minutes), transfer the supernatant to the adsorption column, centrifuge at 12,000 rpm for 1 minute, and discard the waste.

5. Add 600 μl Buffer PW to the column and centrifuge at 12,000 rpm for 1 minute to remove the waste.

6. Repeat the above steps.

7. Open for 2 minutes.

8. Place in an oven at 55 ° C for 20 minutes.

9. Place the column in a new centrifuge tube and add 50 μl of preheated ddH2O to the middle of the membrane for 2 minutes at room temperature and 1 minute at 12,000 rpm. The resulting solution was re-added to the adsorption column, allowed to stand at room temperature for 2 minutes, and centrifuged at 12,000 rpm for 1 minute.

 

PGL3 series Plasmid transfection

1. Plate the cells one day prior to transfection and switch to antibiotic-free 10% FBS DMEM medium and place 1X105 per well in 24-well plates. According to PGL3-Basic, PGL3-Hulc, PGL3-hTERT, PGL3-Control four groups, each group has four duplicate wells, totaling 16 wells.

2. Dilute Lipofectamine 3000 reagent in opti-MEM medium, 1.5 μL Lipofectamine 3000 per tube. Therefore, the medium 25*38 = 950 μl and Lipofectamine 3000 1.5*38 = 57 μl of a total of 1007 μl of the solution were uniformly mixed.

3. Dilute P3000 reagent in opti-MEM medium, prepare according to 2μl/μg plasmid, and mix each group with pRL-tk plasmid in a 10:1 relationship, ie 450ng+45ng/well, to ensure the total amount is 500 ng / hole. Therefore, there are the following systems:

(1) PGL3-Basic: medium 25*8=200 μl Basic plasmid 17 μl pRL-tk plasmid 1 μl P3000 1*8=8 μl

(2) For PGL3-Hulc: medium 25*8=200 μl hulc plasmid 11 μl pRL-tk plasmid 1 μl P3000 1*8=8 μl

(3) For PGL3-hTERT: medium 25*8=200 μl hTERT plasmid 12 μl pRL-tk plasmid 1 μl P3000 1*8=8 μl

(4) For PGL3-control: medium 25*8=200 μl Control plasmid 5.7 μl pRL-tk plasmid 1 μl P3000 1*8=8 μl

4. Mix the solutions in steps 2 and 3 uniformly (200 μl plus 200 μl) for 15 minutes, then add 50 μl per well to the 950 μl opti-MEM medium/ The wells were incubated in a 24-well plate in a 37 ° C 5% CO 2 incubator.

5. After 6H, switch to 10% DMEM medium and continue to culture, and test after 36h.

 

Preparation of DH5α competent state (transformation)

1. Take 200 μl of the strain stored at -20 °C and add it to 20 ml of LB medium, and incubate at 37 °C, 220 r/min shaker overnight.

2. The next day, 200 μl of the overnight culture solution was transferred to 20 ml LB, and cultured at 37 °C, 220 r/min for 2 h.

3. Pipette 1.5 ml into the centrifuge tube (8 in total) and centrifuge at 5800 rpm for 5 min at 4 °C.

4. Discard the supernatant in the clean bench, collect the bacterial pellet, add 600 μl of pre-cooled 0.1 mol/L CaCl 2 solution, and gently pipette to uniformly disperse the cells, and ice bath for 30 min.

5. Centrifuge at 5,800 rpm for 5 min at 4 °C.

6. Discard the supernatant in the clean bench, collect the bacterial pellet, add 100 ul of pre-cooled 0.1 mol/L CaCl2 solution, gently blow to make the cells evenly dispersed, and store at 4 °C for use, a total of 8 sticks.

 

Preparation of DH5α competent state (transformation)

1. Take 200 μl of the strain stored at -20 °C and add it to 20 ml of LB medium, and incubate at 37 °C, 220 r/min shaker overnight.

2. The next day, 200 μl of the overnight culture solution was transferred to 20 ml LB, and cultured at 37 °C, 220 r/min for 2 h.

3. Pipette 1.5 ml into the centrifuge tube (8 in total) and centrifuge at 5800 rpm for 5 min at 4 °C.

4. Discard the supernatant in the clean bench, collect the bacterial pellet, add 600 μl of pre-cooled 0.1 mol/L CaCl 2 solution, and gently pipette to uniformly disperse the cells, and ice bath for 30 min.

5. Centrifuge at 5,800 rpm for 5 min at 4 °C.

6. Discard the supernatant in the clean bench, collect the bacterial pellet, add 100 ul of pre-cooled 0.1 mol/L CaCl2 solution, gently blow to make the cells evenly dispersed, and store at 4 °C for use, a total of 8 sticks.

 

pcDNA6.2-hTERT double enzyme digestion

1. Preparation of 2% nucleic acid electrophoresis gel: Weigh 1g agar, add it to 50mL of 1xTAE, mix and dissolve in microwave oven for about 2min. After a little cooling, 5 uL of golden view dye was added, poured into a rubberized plate, and gelled after about 25 minutes.

2. Double digestion of the vector pcDNA6.2-hTERT fragment with restriction endonucleases SpeI-HF and SacI-HF

 

Cell recovery

1. Take a 15 mL centrifuge tube and add 9 mL of serum-free medium.

2. The cells were removed from the -80 ° C freezer and thawed in a 37 ° C water bath (within 1 min).

3. After wiping with a 75% ethanol cotton ball, the cell cryotube was opened and transferred to a centrifuge tube.

4. Centrifuge for 5 min at 1000 rpm and discard the supernatant.

5. Add 1 mL of complete medium to the centrifuge tube, pipette until it is dispersed into a single cell suspension, transfer to a cell culture flask, and add 3 mL of complete medium.

6. Incubate at 37 ° C, 5% CO2.

7. After 24 h, the medium was changed according to the cell growth state.

 

Cell inheritance

1. When the cell culture density is greater than 80%, passage can be carried out.

2. The medium in the flask was discarded and 1 mL of trypsin was added for rinsing to remove residual medium.

3. Add 1 mL of trypsin, gently shake the trypsin to infiltrate the cell bottle, place at 37 °C, digest for 1 min 30 s.

4. Under the microscope, when the cell morphology became round, it showed complete digestion.

5. Add 2 mL of medium directly to stop digestion (this step should be done quickly to avoid overdigestion).

6. The digested cells were transferred to a T75 flask and 9 mL of medium containing 10% FBS was added.

7. Incubate at 37 ° C, 5% CO2.

8. After 24 h, the medium was changed according to the cell growth state.