Notebook

Notebook

7.8

1. The PCR system was formulated according to the table below (YFP)

2. Put in PCR instrument, PCR program

3. Agarose gel electrophoresis

1. Weigh 0.5g agarose and add to 50ml 1X TAE
2. Microwave until completely melted
3. Cool to 50 °C to 60 °C and add 3μL DNA stain
4. Inverted board
5. After cooling, sample in sequence
6. 130V 30min
7. After the process is completed, place the gel under a UV lamp, excise the portion of the gel containing the target fragment, and place the excised gel into a 1.5 mL EP tube.

7.9

1. Gel recovery

1. Add 200 μL of PN to the colloid and incubate in a water bath at 50°C for 10 minutes.
2. Add 500 μL of BL to the chromatography column and place it in a centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
3. Transfer the liquid in the EP tube to the chromatography column and let it stand at room temperature for 2 minutes. Then place it in the centrifuge at 12,000 rpm for 1 minute and discard the eluate.
4. Add 600 μL of PW to the column and place it in the centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
5. Repeat the previous step once.
6. Place the tube in the centrifuge at 12,000 rpm for 2 minutes without any liquid, then open the lid and let it stand for 5 minutes.
7. Place the chromatography column in a new EP tube and add 30 μL of ddH2O to the column. Let it stand for 2 minutes and then place it in the centrifuge at 12,000 rpm for 2 minutes. Collect the eluate.
8. Add the eluate back to the chromatography column and place the system in the centrifuge at 12,000 rpm for 2 minutes. Discard the chromatography column and keep the EP tube.

2. Enzymatic digestion

1) Prepare the enzyme digestion system

2) 37℃ 40min

3. Agarose gel electrophoresis + Gel recovery

1. Weigh 0.5g agarose and add to 50ml 1X TAE
2. Microwave until completely melted
3. Cool to 50 °C to 60 °C and add 3μL DNA stain
4. Inverted board
5. After cooling, sample in sequence
6. 130V 30min
7. After the process is completed, place the gel under a UV lamp, excise the portion of the gel containing the target fragment, and place the excised gel into a 1.5 mL EP tube.
8. Add 200 μL of PN to the colloid and incubate in a water bath at 50°C for 10 minutes.
9. Add 500 μL of BL to the chromatography column and place it in a centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
10. Transfer the liquid in the EP tube to the chromatography column and let it stand at room temperature for 2 minutes. Then place it in the centrifuge at 12,000 rpm for 1 minute and discard the eluate.
11. Add 600 μL of PW to the column and place it in the centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
12. Repeat the previous step once.
13. Place the tube in the centrifuge at 12,000 rpm for 2 minutes without any liquid, then open the lid and let it stand for 5 minutes.
14. Place the chromatography column in a new EP tube and add 30 μL of ddH2O to the column. Let it stand for 2 minutes and then place it in the centrifuge at 12,000 rpm for 2 minutes. Collect the eluate.
15. Add the eluate back to the chromatography column and place the system in the centrifuge at 12,000 rpm for 2 minutes. Discard the chromatography column and keep the EP tube.

4. The connection of the YFP and pRSETa vectors

1）Prepare the connection system（10μL）

2）4℃ for the whole night

7.10

1． Transformation of the connected product

1. Add 10 μL of plasmid to the EP tube.
2. Add 20 μL of competent cells to the EP tube.
3. Place the system on ice and let it stand for 30 minutes.
4. After completion, quickly transfer the system to a water bath and perform a heat shock at 42°C for 35 seconds.
5. Add 500 μL of LB culture medium to the system and place the entire system in a shaker for 1 hour.
6. After the previous section is completed, place the entire reaction system on the centrifuge and centrifuge it at 4000 rpm for 5 minutes.
7. After completion, discard the supernatant of the system, 450 μL, and then use the remaining liquid to resuspend and mix the precipitate before coating the plate.

2. Amplification of the mCherry fragment

1） The PCR system was formulated according to the table below（mCherry）

2）．Put in PCR instrument, PCR program

Cycle：35

7.11

1. Perform single-cell cloning culture and identification-YFP

1. Add 500 μL LB culture medium to a 2 mL centrifuge tube
2. Pick a single colony into the tube
3. Place the system in a shaking incubator at 37°C and 220 rpm for culture until the OD value reaches 0.5
4. Perform PCR identification

2. Send the sequenced strains for further sequencing and identification.

7.14

1. Gel recovery(mCherry)

1. Add 200 μL of PN to the colloid and incubate in a water bath at 50°C for 10 minutes.
2. Add 500 μL of BL to the chromatography column and place it in a centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
3. Transfer the liquid in the EP tube to the chromatography column and let it stand at room temperature for 2 minutes. Then place it in the centrifuge at 12,000 rpm for 1 minute and discard the eluate.
4. Add 600 μL of PW to the column and place it in the centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
5. Repeat the previous step once.
6. Place the tube in the centrifuge at 12,000 rpm for 2 minutes without any liquid, then open the lid and let it stand for 5 minutes.
7. Place the chromatography column in a new EP tube and add 30 μL of ddH2O to the column. Let it stand for 2 minutes and then place it in the centrifuge at 12,000 rpm for 2 minutes. Collect the eluate.
8. Add the eluate back to the chromatography column and place the system in the centrifuge at 12,000 rpm for 2 minutes. Discard the chromatography column and keep the EP tube.

2. Isolation and cultivation of positive clones of E. coli (YFP)

1. Add 5mL LB and 5 μL Amp to a 50 mL centrifuge tube, and then add the positive cloning bacterial solution that was saved last week.
2. Shake at 37℃ and 220 rpm for over night.

7.15

1. Plasmid extraction

1. Take 5ml overnight culture bacterial solution, centrifuge at 9000 rpm for 2 minutes, and collect all bacterial bodies after multiple centrifugation
2. The supernatant was removed and 250μL buffer P1 was added to the precipitation
3. Blow the precipitation repeatedly with the pipette gun until the bacteria are completely suspended
4. Add 250μL Buffer P2, and immediately gently reverse the centrifugal tube for 5-10 times and mix well. Let stand at room temperature for 2-4 minutes. You'll see the liquid clear and clear
5. Add 300μL Buffer P3 and immediately gently reverse the centrifuge tube for 5-10 times to mix. At this point you can see the white precipitate forming
6. Centrifuge at 12000rpm for 5-10 min. Transfer the supernatant into the adsorption column, centrifuge at 12000rpm for 30 seconds, and drain the liquid in the collection tube. If the supernatant liquid is too much, it can be centrifuged
7. Add 600μL Wash Solution, centrifuge at 9000rpm for 30 seconds, and drain the liquid in the collection tube. Repeat twice
8. The empty adsorption column is placed in the collection tube at 9000rpm for 2min
9. Place the adsorption column in a new centrifuge tube and place it in the fume hood for 10min to remove residual ethanol
10. Add 30μL Elution Buffer into the adsorption column, leave at room temperature for 2min, and then centrifuge at 9000rpm for 2min to collect the liquid in the centrifugal tube
11. In order to ensure the concentration of glue recovery, you can repeat the step j)

2. Send the purified plasmid for sequencing

3. Enzymatic digestion

Prepare the enzyme digestion system

37℃ 40min

3. Agarose gel electrophoresis+ Gel recovery

1. Weigh 0.5g agarose and add to 50ml 1X TAE
2. Microwave until completely melted
3. Cool to 50 ° C to 60 ° C and add 3μL DNA stain
4. Inverted board
5. After cooling, sample in sequence
6. 130V 30min
7. After the process is completed, place the gel under a UV lamp, excise the portion of the gel containing the target fragment, and place the excised gel into a 1.5 mL EP tube.
8. Add 200 μL of PN to the colloid and incubate in a water bath at 50°C for 10 minutes.
9. Add 500 μL of BL to the chromatography column and place it in a centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
10. Transfer the liquid in the EP tube to the chromatography column and let it stand at room temperature for 2 minutes. Then place it in the centrifuge at 12,000 rpm for 1 minute and discard the eluate.
11. Add 600 μL of PW to the column and place it in the centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
12. Repeat the previous step once.
13. Place the tube in the centrifuge at 12,000 rpm for 2 minutes without any liquid, then open the lid and let it stand for 5 minutes.
14. Place the chromatography column in a new EP tube and add 30 μL of ddH2O to the column. Let it stand for 2 minutes and then place it in the centrifuge at 12,000 rpm for 2 minutes. Collect the eluate.
15. Add the eluate back to the chromatography column and place the system in the centrifuge at 12,000 rpm for 2 minutes. Discard the chromatography column and keep the EP tube.

4. The connection of the mCherry and pRSETa vectors

1）Prepare the connection system（10μL）

2）4℃ for the whole night

7.16

1． Transformation of the connected product

1. Add 10 μL of plasmid to the EP tube.
2. Add 20 μL of competent cells to the EP tube.
3. Place the system on ice and let it stand for 30 minutes.
4. After completion, quickly transfer the system to a water bath and perform a heat shock at 42°C for 35 seconds.
5. Add 500 μL of LB culture medium to the system and place the entire system in a shaker for 1 hour.
6. After the previous section is completed, place the entire reaction system on the centrifuge and centrifuge it at 4000 rpm for 5 minutes.
7. After completion, discard the supernatant of the system, 450 μL, and then use the remaining liquid to resuspend and mix the precipitate before coating the plate.

7.17

1. Monoclonal culture

1. Add 1 mL of LB medium and 1 μL of Amp (100 mg/mL) into a 1.5 mL EP tube.
2. Pick a single colony from the overnight grown solid medium of Escherichia coli and add it to the above 1.5 mL EP tube.
3. Shake at 37°C and 220 rpm until the bacterial solution becomes turbid.

2. Bacterial liquid PCR identification

The clarified bacterial solution obtained above was used as the template for PCR analysis. The PCR system was based on the PCR amplification of mCherry.

7.18

1. Isolation and cultivation of positive clones of E. coli (mCherry)

1. Add 5mL LB and 5 μL Amp to a 50 mL centrifuge tube, and then add the positive cloning bacterial solution that was saved last week.
2. Shake at 37℃ and 220 rpm for over night.

2. Plasmid extraction

The process refers to the plasmid extraction method of YFP.

7.21

1. PCR

Using different primers to perform PCR amplification on the sequence of YFP, different lengths of Linker were constructed. The PCR system followed the amplification of YFP, and the primer sequences are as follows.

2. Send the extracted mCherry plasmid for sequence analysis

3. Gel recovery （sequences of N1-N3 PCR）

7.22

1. Enzymatic digestion: DpnI enzyme digests the gel recovery product and the vector at 37°C for 40 minutes.
2. Ligation: T4 DNA ligase ligates the above-mentioned ligated products at 4°C for 2 hours.
3. Transformation: The above-mentioned ligated products are transformed into Escherichia coli competent cells. The experimental procedure follows the previous one.

7.23

1. PCR

Using different primers to perform PCR amplification on the sequence of YFP, different lengths of Linker were constructed. The PCR system followed the amplification of YFP, and the primer sequences are as follows.

2. Monoclonal culture: Select the monoclonal cells grown on the solid medium after transformation on July 22nd and cultivate them at 37°C with a speed of 220 rpm.

7.24

1. Plasmid extraction

The process refers to the plasmid extraction method of YFP.

2. Send the extracted plasmid for sequencing

3. Gel recovery：Recycle the PCR product with a concentration of 7.23

7.25

1. Enzymatic digestion: DpnI enzyme digests the gel recovery product and the vector at 37°C for 40 minutes.
2. Ligation: T4 DNA ligase ligates the above-mentioned ligated products at 4°C for 2 hours.
3. Transformation: The above-mentioned ligated products are transformed into Escherichia coli competent cells. The experimental procedure follows the previous one.

7.28

1. transformation: Transform the sequenced correct YFP plasmid into BL21 competent cells.
2. Pick and cultivate the cloned colonies obtained from the 7.25 transformation of E. coli.

7.29

1. Protein induction: Induce the BL21 strain containing the YFP plasmid with IPTG. The induction conditions are 18°C, 220 rpm, and 24 hours.
2. Plasmid extraction
3. The process refers to the plasmid extraction method of YFP.
4. Send the extracted plasmid for sequencing

7.30

1. The cultivation of E. coli

1. Add 20 microliters of Amp
2. Add 20 milliliters of LB
3. Add 20 microliters of bacterial solution
4. Shaking culture at 37℃ and 220 rpm on a shaker

2. transformation

1. Take the competent cells out from the -80°C refrigerator.
2. Add 1 μL of plasmid and 4-5 μL of competent cells to the EP tube.
3. Incubate on ice for 30 minutes, then in a water bath at 42°C for 35 seconds.
4. Add 500 μL of LB in the ultrapure bench, incubate at 37°C, on a 220 rpm shaker for 1 hour. During this period, sterilize the inoculation rod with an alcohol lamp.
5. Centrifuge at 4000 rpm for 5 minutes, discard 450 μL of the supernatant, retain 100 μL of the precipitate and the supernatant. Take 100 μL and evenly drop onto the petri dish, spread, and finish by placing upside down and sealing.
6. Cultivate in an inverted 37°C incubator overnight.

7.31 Purification of NorR-YFP probe protein

1. Check whether there is fluorescence in the overnight-induced E. coli culture medium.
2. Centrifuge E. coli for 5 minutes at 5000 rpm
3. Discard the upper layer of water
4. Add 1 ml of PBS and vortex to mix evenly
5. Transfer to a 2 ml tube
6. Use diaphragm meter + magnetic beads
7. Centrifuge for 10 minutes at 12,000 rpm
8. Discard the upper clear liquid.
9. Add the egg white to the magnetic beads.
10. Place it in the protein purification apparatus for 3 hours.
11. Discard the upper clear liquid.
12. Add 500ml of 50kpi and shake to discard the upper clear liquid.
13. Add 500ml of 100kpi and shake to retain the upper clear liquid.
14. Add 500ml of 300kpi and shake to retain the upper clear liquid.

8.1

1. Fluorescent protein spectroscopy - Analysis of the full spectrum (300 - 800)

1. Wash the quartz glass dish.
2. Add 200 μL of YFP fluorescent protein.
3. Place the sample in the instrument.
4. Set the corresponding program and start the test.
5. Record the data.
6. Test completed and output results

2. Detect the specificity of fluorescent proteins

8.4

The influence of temperature and pH on the probe

1. Prepare solutions of different pH using NaOH solution and HCI solution.
2. Add 90 μL of solutions with pH values of 7, 7.4, 7.5, and 7.6 to a 96-well plate (7 groups for each treatment, totaling 28 wells). Add 10 μL of protein probe solution to each well. After adding the protein, place the plate in the microplate reader for detection at 37°C for 20 times. After testing 5 groups, add 10 μL of furazolidone to each well.

8.5

mCherry E. coli cloning culture

Pick and transfer 500 μL of LB medium + 0.5 μL Amp + the monoclonal bacteria

8.6

1. PCR（GFP）

Tm：55℃，cycle：35

2. Agarose gel electrophoresis and gel recovery

1. Run the gel: Inject 5 μL of the marker into the first well of the gel, and all the amplified products into the remaining wells of the gel.
2. Gel recovery: #PN actually adds 200 μL per tube, PW actually adds 500 μL per tube. Place for 5 minutes in the seventh step, and do not use EB but use 30 μL of ddH2O in the eighth step.

8.7

1. Induction of mCherry protein expression

The bacterial culture containing mCherry was induced with IPTG at low temperature (18℃, 220 rpm) for 24 hours, then left at room temperature for 30 minutes, and then centrifuged at 4000 rpm for 5 minutes to break the bacteria and release the protein.

2. Protein extraction and detection of protein's response to NO

Discard the supernatant, add 1 mL of HEPES buffer, vortex to detach the protein from the tube wall, and add 2 mL into the EP tube

3. Protein disruption

(instrument needs repair, will be done next week)

4. Gel recovery

Gel recovery: #PN actually adds 200 μL per tube, PW actually adds 500 μL per tube. Place for 5 minutes in the seventh step, and do not use EB but use 30 μL of ddH2O in the eighth step.

5. The connection of the GFP and pRSETa vectors

1）Prepare the connection system（10μL）

2）4℃ for the whole night

8.8

Transformation of the connected product

1. Add 10 μL of connected product to the EP tube.
2. Add 20 μL of competent cells to the EP tube.
3. Place the system on ice and let it stand for 30 minutes.
4. After completion, quickly transfer the system to a water bath and perform a heat shock at 42°C for 35 seconds.
5. Add 500 μL of LB culture medium to the system and place the entire system in a shaker for 1 hour.
6. After the previous section is completed, place the entire reaction system on the centrifuge and centrifuge it at 4000 rpm for 5 minutes.
7. After completion, discard the supernatant of the system, 450 μL, and then use the remaining liquid to resuspend and mix the precipitate before coating the plate.

8.11

1. Pick a single clone [Expand the bacterial quantity]

500 μL LB + 0.5 μL Amp + single clone

2. Serum detection (test probe)

Artificial mouse plasma was divided into four portions (50 μL each), and 0, 300 μmol, 500 μmol, 1 mmol, and 1.5 mmol of furazan were added respectively. Then, 50 μL of probe solution was added to each portion, and the mixtures were observed under a blue light instrument. The final volumes of the five groups were 100, 121.4, 150, 105.6, and 108.8 μL, respectively.

3. Recovery of 293T engineering cells:

Remove the cells from the -80℃ refrigerator and quickly place them in a water bath at 37℃.

2 mL of DMEM medium (containing 10% fetal bovine serum) + one tube of frozen cells, 1000 rpm (but in actual operation, it was set to 1000 * g) for 3 minutes, discard the supernatant (the cryopreservation solution is toxic to the cells), add 2 mL of DMEM and vortex, add to the petri dish, then add 6 mL of DMEM.

冻存的细胞

8.12

1. Plasmid extraction: Take 2 mL of bacterial solution and transfer it to a 2 mL ELP tube. Centrifuge at 12000 rpm for 1 min * 3
2. 293T cell passage

Parental cells before passage

Place 6 cell cards into the six-well plate respectively, add 2 mL of DMEM to each well, add 8 mL of DMEM to the empty wells, discard all the supernatant from the previous culture plates, and add 1 mL of trypsin to cause the cells to detach.

Rinse off the remaining cells with pancreatic enzyme solution, resuspend, and transfer to a 15 mL tube. Centrifuge at 1000 rpm for 3 minutes, discard the supernatant, add 2 ml of new medium, resuspend. Add 500 microliters to a petri dish and mix thoroughly in a cross pattern [for preservation]. Add 150 microliters per well to a six-well plate.

8.13

1. Transfection

   The final shape of the cell

   The optimal density of cells

   Add 80 microliters of DMEM (without serum) to each of the 6 pear-shaped tubes, and add 2 microliters of PIvX-NO {cyto,mito,nuclear} (with volumes of {2.53, 2.31, 2.61 microliters}) to {2x3} of the tubes. Add 36 microliters of EZtrans 480 microliters of DMEM (without serum) to tube 7, and distribute it into tubes 1-6 in six portions and vortex. Let it stand for 10 minutes.

   Replace the liquid in the six-well culture medium with 2 mL of DMEM (without serum)

   Add the solutions after standing to the six-well plates respectively

   The transfected cells

2. Transformation(mCherry-BL21)

8.14

1. Single-cell cloning (mCherry-BL21)
2. Passage of 293T cells
3. Observation of fluorescence
4. Bacterial inoculation: Add bacterial culture solution (20 mL), LB medium, and 20 microliters of Amp to a 50 mL centrifuge tube

8.15

1. 293T cell passage
2. Confocal imaging: Attach the thin slices containing 293T cells that have been transfected for 48 hours onto the confocal carrier, and rinse with 500 μL of sugar-free HBSS.