Each month is depicted with a grid system where each grid corresponds to a 3-day period. The shaded regions indicate the days designated for work.

January：

February：

March：

April：

May：

June：

July:

August:

September:

October:

Pre-experiment safety training：

The main content is as follows：

I. Always wear required PPE: lab coat, gloves, and safety goggles in the laboratory.

II. Know locations and operations of emergency equipment (eyewash stations, fire extinguishers, safety showers).

III. Never consume food/beverages or apply cosmetics in lab areas.

IV. Follow proper handling/disposal protocols for chemicals, biohazards, and sharps.

V. Maintain clean, organized workspaces to prevent accidents and contamination.

VI. Immediately report spills, accidents, or injuries to the supervisor.

VII. Perform only authorized, trained procedures under supervision.

VIII. Operate equipment strictly per designated functions and safety protocols.

IX. Memorize emergency procedures including evacuation routes and assembly points.

X. Attend regular safety training and comply with all guidelines to ensure workplace safety.

Part 1: Plasmid Extraction

Purpose

To extract and purify plasmid DNA from cultured bacteria for use in downstream applications such as PCR.

Procedure

1. Bacterial Collection
   • Transfer 1.5 mL of bacterial culture into a centrifuge tube.
   • Centrifuge at 10,000 × g for 1 minute at room temperature.
   • Discard the supernatant.
2. Cell Resuspension
   • Add 250 μL of Solution A.
   • Resuspend the pellet completely.
3. Cell Lysis
   • Add 250 μL of Solution B.
   • Gently invert 4–6 times.
   • Let stand for 2 minutes to lyse cells.
4. Protein and Debris Precipitation
   • Add 350 μL of Solution C.
   • Gently invert until white precipitates form.
   • Centrifuge at 10,000 × g for 10 minutes at room temperature.
5. DNA Binding
   • Transfer the supernatant to a new tube containing the DNA adsorption column.
   • Centrifuge at 10,000 × g for 1 minute.
6. Protein Removal
   • Discard the filtrate.
   • Add 500 μL Buffer HB.
   • Centrifuge for 1 minute at 10,000 × g.
7. Column Washing (x2)
   • Add 750 μL Wash Buffer, centrifuge for 1 minute.
   • Discard filtrate.
   • Repeat with another 750 μL Wash Buffer and centrifuge again.
8. Column Drying
   • Centrifuge the empty column at 10,000 × g for 1 minute to remove ethanol.
9. DNA Elution
   • Place column into a clean tube.
   • Add 50 μL TE Buffer to the center of the membrane.
   • Let sit 2–5 minutes.
   • Centrifuge at 10,000 × g for 1–5 minutes.
   • Store eluted DNA at –20°C.
10. DNA Quantification

• Measure concentration and purity of eluted plasmid using a Nanodrop.

Part 2: Agarose Gel Electrophoresis, Gel Extraction, Seamless Cloning, and Transformation

Purpose

To confirm the success of PCR by performing agarose gel electrophoresis and visualizing DNA bands. Target DNA fragments are then extracted and purified from the gel for use in seamless cloning. The final recombinant plasmid is transformed into competent E. coli cells to enable propagation for further applications.

Procedure

1. Agarose Gel Preparation and Electrophoresis

• Mix 2 g agarose with 100 mL of TAE buffer and water (1:1 ratio).
• Microwave until agarose is fully dissolved.
• Cool the solution slightly and add YeaRed nucleic acid dye.
• Pour the gel into a mold with a comb and allow to solidify completely.
• Place the gel into the electrophoresis tank and cover with TAE buffer.
• Mix each DNA sample with loading buffer at a 1:5 ratio.
• Load DNA samples and a DNA Marker into the wells.
• Run electrophoresis at 180 V for 20 minutes.
• Visualize the DNA bands using a gel documentation system.
• Identify and record the size of DNA fragments by comparing to the Marker.

2. DNA Gel Extraction and Purification

• Excise the desired DNA band from the gel using a clean blade.
• Melt the gel slice and transfer it into a filter column.
• Centrifuge at 8000 rpm for 60 seconds, discard flowthrough.
• Repeat filtration and centrifugation to remove gel debris.
• Wash the filter column twice with washing buffer, centrifuging each time at 8000 rpm for 60 sec.
• Open the column lid and centrifuge once more to remove residual ethanol.
• Transfer the column to a clean Eppendorf tube.
• Add elution buffer, incubate briefly, and centrifuge to collect purified DNA.
• Use a Nanodrop or spectrophotometer to measure DNA concentration.

3. Seamless Cloning Reaction

• In a tube, mix:
• Target fragment 1 (at 0.04 × its length, e.g., 36 ng)
• Target fragment 2 (at 0.02 × its length, e.g., 180 ng)
• 5× recombination buffer
• Homologous recombinase (enzyme:DNA ratio = 1:10)
• Incubate the mixture at 37°C for 30 minutes to allow ligation into a circular plasmid.

4. Transformation into Competent Cells

• Thaw competent E. coli cells on ice.
• Add recombinant plasmid mixture to cells and incubate on ice for 30 minutes.
• Heat shocks the mixture at 42°C for 45 seconds.
• Immediately chill on ice for 2 minutes.
• Add LB medium and incubate at 37°C, 220 rpm for 1 hour to allow recovery.
• Plate the bacteria on LB agar plates.
• Incubate plates overnight at 37°C for colony growth.
• Perform screening to identify positive transformants.

Part 3: Colony PCR, Gel Electrophoresis, and Strain Preservation

Purpose

To confirm successful bacterial transformation by performing PCR on selected colonies. Then, to visualize DNA bands through gel electrophoresis and preserve validated bacterial strains for future use.

Procedure

1. Colony Selection and Preservation

• Pick individual bacterial colonies from transformation plates.
• Streak colonies onto new agar plates for backup.
• Inoculate selected colonies into LB broth with corresponding antibiotic.
• Shake at 37°C, 220 rpm to allow bacterial expansion.

2. PCR Setup

• In a microtube, prepare the PCR reaction:
  • Bacterial colony (as DNA template)
  • PCR buffer
  • Forward and reverse primers
  • dNTPs
  • DNA polymerase
  • Sterile distilled water
• Briefly centrifuge to collect the solution at the bottom.

3. PCR Amplification

• Denature DNA at 95°C to separate strands.
• Anneal primers at approximately 50°C to bind the template.
• Extend DNA strands at 72°C using Taq polymerase and dNTPs.
• Store PCR products at 4°C or –20°C until analysis.

4. Agarose Gel Electrophoresis

• Prepare 1% agarose gel using TAE buffer (0.5× or 1×).
• Heat the agarose in a microwave until fully dissolved.
• Cool to ~60°C, add DNA stain (1:10,000), and mix well.
• Pour into gel tray with comb and allow to solidify.
• Mix PCR products with loading dye (1:5 ratio).
• Load DNA samples and DNA Marker into the gel wells.
• Submerge gel in TAE buffer until just covered.
• Connect the electrodes (sample wells near the negative pole).
• Run electrophoresis at appropriate voltage (e.g., 100–180 V).
• Visualize bands under UV using a gel imager.

5. Data Analysis and Strain Backup

• Analyze the gel image to determine PCR success based on band size.
• Identify and mark successful transformants.
• Preserve confirmed positive colonies by streaking or freezing for long-term use.

Part 4 Verification

Purpose:
To verify successful ligation and expression by plasmid extraction and electrophoresis, and to continue culturing positive bacterial strains for sequencing validation.

Procedures:

• Inoculate single colonies into LB liquid medium with antibiotics in 12 mL culture tubes.
• Shake at 37°C, 220 rpm overnight.
• Transfer 1.5 mL of each culture to centrifuge tubes, spin down at 10,000 × g for 1 min.
• Perform plasmid extraction using commercial kit:
  • Resuspend in Solution A
  • Lyse with Solution B
  • Neutralize with Solution C
  • Bind DNA to column
  • Wash twice with wash buffer
  • Elute with TE buffer
• Measure plasmid concentration with Nanodrop.
• Prepare 1% agarose gel with TAE buffer and nucleic acid dye.
• Load plasmid DNA with loading dye and run electrophoresis with DNA Marker.
• Visualize bands under gel imager to confirm presence and size of plasmid.
• Store extracted plasmids at –20°C for sequencing.
• Preserve successful bacterial cultures by streaking on new plates or freezing glycerol stocks.

Part 5 SDS-PAGE

Purpose:
To extract and purify a specific target protein (e.g., PD-1) from bacterial lysate.

Procedures:

1. Protein Extraction

• Add lysozyme and lysis buffer to bacterial pellet. Mix until fully resuspended.
• Place tube in an insulated container with ice. Sonicate at 50% power (200–300 W) with 5-second intervals, total of 6 minutes.
• Centrifuge lysate at 1000 × g for 10 seconds at 4°C. Collect supernatant (crude protein extract).
• Prepare column in parallel by resuspending nickel affinity gel and equilibrating it with lysis buffer at 4°C.

2. Protein Purification

• Mix crude extract with equilibrated resin. Shake at 4°C for 1.5 hours.
• Load mixture into gravity column. Let drain slowly and repeat 3 times. Backup samples.
• Wash with washing buffer and collect flow-through (wash fractions).
• Elute protein with elution buffer. Collect elution fractions and label clearly.

3. SDS-PAGE Validation

• Assemble gel plates. Pour separating gel, level with ethanol, let set. Pour stacking gel and insert comb.
• Assemble gel into electrophoresis apparatus. Fill with Tris-Glycine-SDS buffer.
• Load samples: elution fractions, wash fractions, crude lysate, and protein ladder.
• Run at 80 V for 25 minutes, then 180 V for 35 minutes (~1 hour total).
• Stain gel and shake for 50 minutes. Image to confirm target protein (e.g., PD-1).

Part 6 Transfer

Purpose:
To transfer and detect specific proteins using SDS-PAGE and PVDF membrane blotting.

Procedures:

1: SDS-PAGE

• Prepare and pour separating gel, level with ethanol, let polymerize. Pour stacking gel and insert comb.
• Mix Tris-Glycine-SDS buffer. Fill electrophoresis tank.
• Mix protein samples with loading buffer. Load samples and ladder.
• Run at 80 V for 25 min, then 180 V for 35 min (total ~1 hour).

2: Protein Transfer

• Cut off stacking gel. Soak PVDF in methanol for 5 minutes.
• Soak all blotting materials in transfer buffer.
• Assemble sandwich: sponge → filter paper → gel → PVDF → filter paper → sponge.
• Place sandwich in transfer tank with buffer and ice packs.
• Transfer at 150 mA constant current for 2 hours.

Part 7 WB

Purpose:
To validate the expression of His-tagged proteins (e.g., PD-1/PD-L1) using SDS-PAGE and Western blot with HRP detection.

Procedures:

1. SDS-PAGE

• Lyse cells using lysis buffer and ultrasonic disruption. Centrifuge and collect protein.
• Mix with SDS loading buffer, heat to denature.
• Load into SDS-PAGE gel with marker.
• Fill tank with running buffer. Run 80 V for 25 min, then 180 V for 35 min.
• Prepare gel for transfer.

2. Western Blot Transfer

• Cut off stacking gel, soak PVDF in methanol.
• Soak all layers in transfer buffer.
• Assemble sandwich: sponge → filter paper → gel → membrane → filter paper → sponge.
• Place in transfer tank with buffer and ice. Run at 150 mA for 2 hours.

3. Blocking and Detection

• Block with 5% BSA for 1 hour at room temp.
• Wash with PBST 3–5 times.
• Incubate with Anti-His-HRP (1:3000) for 2 hours.
• Wash with PBST 5 times.
• Add TMB substrate. Observe color.
• Image using gel imager.

Part 8 ELISA: Antibody Titer Validation

Purpose:
To verify the titer and binding activity of antibodies specific to PD-1/PD-L1.

Procedure:

1. Coat wells with PD-1/PD-L1 proteins. Incubate at 4°C overnight.
2. Wash 3x with PBST. Block with 3% BSA for 1 hour. Wash 2x.
3. Add Anti-His HRP antibody, incubate 2 hours. Wash 3x.
4. Add TMB, incubate 20 minutes in dark. Add stop solution.
5. Measure absorbance at 650 nm with microplate reader.