Experiments

Experiments: We learned the following techniques and experiments in our school laboratory from Mr. Gert Grobler: 1) To use Micropipettes: This involves accurately measuring and transferring small volumes of liquids, essential for precise lab work in molecular biology experiments. 2) Gel Electrophoresis: A technique used to separate DNA based on their size and charge by applying an electric current through a gel matrix. 3) Making Agar Plates: This process involves preparing nutrient-rich agar media in petri dishes, which serves as a growth environment for microorganisms or cells. 4) To use an Autoclave: The autoclave is used for sterilizing laboratory equipment, media, and waste by applying high-pressure steam to eliminate contaminants and pathogens. 5) To use a DNA Extraction (Kit): This involves using a commercially available kit to isolate and purify DNA from cells or tissues, ensuring high-quality DNA for downstream applications. We use it to extract DNA from aromatic plants. 6) To use a PCR Machine: A PCR (Polymerase Chain Reaction) machine amplifies specific DNA sequences through thermal cycling, enabling the rapid duplication of DNA for analysis. We used it to amplify rbcL (Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene). This gene is found in the chloroplast genome and is widely used in plant DNA barcoding due to its universal presence and slow mutation rate, which makes it useful for identifying plant species at higher taxonomic levels. 7) DNA sequence analysis: We used Chromas and Mega software to analyze the DNA barcode sequences. We used Genebank to identify the species.

We learned the following techniques and experiments from Ms. Je Hou in the laboratory of the Shenzhen Institute of Translational Medicine of The First Affiliated Hospital of Shenzhen University, China: 1) Clean Bench Usage and Cautions: Clean benches provide a sterile workspace with laminar airflow, and users must avoid introducing contaminants by using proper sterilization techniques and keeping materials and movements within the sterile area to a minimum. 2) Growing Bacteria in Shaker: This involves cultivating bacterial cultures in a liquid medium while continuously agitating them to ensure even distribution of nutrients and oxygen for optimal bacterial growth. 3) Growing Bacteria in Incubator: Bacteria are grown in a controlled-temperature incubator, providing the ideal conditions for replication and growth over a set period, usually at 37°C. 4) To use a Gibson Assembly (Kit): This method allows for the seamless joining of multiple DNA fragments by using enzymes to create a contiguous sequence, often used in molecular cloning. 5) To use a Plasmid Extraction (Kit): This process isolates and purifies plasmid DNA from bacterial cells using a commercially available kit, typically involving cell lysis, binding DNA, washing, and elution. 6) To use a Gel Extraction (Kit): This technique is used to extract and purify specific DNA fragments from agarose gels after electrophoresis, often following a PCR or restriction digestion. 7) To use a Nanodrop: A Nanodrop spectrophotometer is used to measure the concentration and purity of DNA by analyzing light absorption at specific wavelengths. 8) To do Chemical Transformation of Sequence/Plasmid into Competent DH5α Cells: In this process, plasmid DNA is introduced into chemically competent DH5α bacterial cells, usually by heat shock, to enable uptake and expression of foreign DNA. 9) Electrical Transformation: Electroporation uses an electric pulse to temporarily permeabilize bacterial cell membranes, allowing plasmid DNA to enter the cells for genetic transformation. 10) Making WB (Western Blot) Solution: Preparing solutions like transfer buffers and blocking agents for Western Blot involves mixing the appropriate chemicals to facilitate protein transfer, antibody binding, and detection. 11) Making TAE Solution: TAE buffer (Tris-Acetate-EDTA) is prepared for use in gel electrophoresis, serving as a conductive medium to separate nucleic acids like DNA or RNA. 12) Making LB Broth: LB (Luria-Bertani) broth is made by dissolving tryptone, yeast extract, and sodium chloride in water, providing nutrients for bacterial growth in liquid cultures. 13) Gel Imaging: Gel imaging involves using UV light or another detection method to visualize DNA bands after electrophoresis, often aided by fluorescent or chromogenic dyes. 14) Preparing Antibiotics: Antibiotics are typically prepared by dissolving a specific concentration of antibiotic powder in sterile water or solvent, then filter-sterilizing and storing for use in bacterial selection experiments. 15) How to Label Different In-Lab Eppendorf’s /Plates/Solutions, etc.: Proper labeling includes indicating sample identity, date, and researcher initials on tubes, plates, and solutions to avoid mix-ups and ensure traceability.

Methods followed in some of the experiments that we learned:

1. Plasmid Extraction (Kit)

• Transfer 1ml of grown LB medium into 1.5ml ependorf
• Centrifuge at 10000×g for 60sec
• Remove LB and leave bacteria precipitates
• Repeat until all bacteria precipitate is collexted at bottom of ependorf
• Add 250ul of Solution I/RNase A, mix thoroughly
• Add 250ul of Solution II, rotate gently to obtain a clear lysate
• Add 350ul of Solution III, immediately invert and mix thoroughly to prevent localized precipitation
• Centrifuge at 13000×g for 10min
• Insert filter column into 2ml collection tube
• Transfer cleared supertant into filter column
• Centrifuge at 13000×g for 60sec
• Discard Filtrate
• Add 500ul HBC buffer
• Centrifuge at 13000×g for 60sec.
• Discard Filtrate
• Add 700ul DNA Wash Buffer
• Centrifuge at 13000×g for 60sec.
• Discard Filtrate
• Add 700ul DNA Wash Buffer
• Centrifuge at 13000×g for 60sec.
• Dry filter column
• Insert filter colum into 1.5ml ependorf
• Add 50ul ddH2O
• Rest at room temperature for 1min
• Centrifuge at 13000×g for 60sec
• Store at -20°C

2. Gibson assembly (Kit)

• 20ul system
• 10ul 2x clone mix
• 0.5ul plasmid template
• ddH2O up to 20ul
• Pipet to mix
• PCR 50°C 50min

3. Chemical translation

• Thaw potent cells on ice
• Add sequence into potent cell in open bench
• Rest on ice for 30min
• Heat in 42°C for 90sec
• Rest on ice for 2min
• Revive in non-antibiotic liquid medium for 40min
• Plate bacteria onto culture medium with corresponding antibiotics
• Grow in incubator

4. DNA barcoding

1. Primers for rbcL Gene Amplification: Forward primer (rbcL-af): 5’-ATGTCACCACAAACAGAGACTAAAGC-3’ -Reverse primer (rbcL-ar): 5’-GTAAAATCAAGTCCACCRCG-3’

2. DNA Extraction:

   • Extract genomic DNA from the plant sample using a plant DNA extraction kit.
   • Check the quality and concentration of the extracted DNA using a Nanodrop spectrophotometer or gel electrophoresis.

3. Prepare PCR Master Mix:

   • In a PCR tube, add the following reagents:
     • 7 µL of plant DNA template
     • 25 µL Master Mix solution (contains PCR buffer, dNTPs and Taq Polymerase)
     • 0.5 µL of 10 µM rbcL-af forward primer
     • 0.5 µL of 10 µM rbcL-ar reverse primer
     • Nuclease-free water to make the final volume up to 50 µL.

4. PCR Cycling Conditions: Program the thermocycler to run the following cycles:

   • Initial denaturation: 94°C for 3 minutes (to denature the DNA)
   • Denaturation: 94°C for 30 seconds (melts the DNA strands)
   • Annealing: 55°C for 30 seconds (allows primers to bind to the rbcL gene)
   • Extension: 72°C for 1 minute (Taq polymerase extends the DNA strand)
   • Repeat for 35 cycles to ensure sufficient amplification.
   • Final extension: 72°C for 5 minutes (to complete extension of all products).

5. PCR Verification via Gel Electrophoresis:

   • Prepare a 1.0% agarose gel with ethidium bromide or another DNA stain.
   • Load 5 µL of PCR product into the gel along with a DNA ladder.
   • Run electrophoresis at 90–100 volts for 45 minutes.
   • Visualize the gel under UV light. The expected band size for the rbcL gene fragment is around 600–700 bp, depending on the plant species.

6. Sequencing (for Barcoding):

• The PCR product was sent to GENEWIZ for sequencing.
• Use the same primers (rbcL-af and rbcL-ar) for sequencing.

7. Data Analysis:
   • Sequences were checked and analyzed with Chromas and MEGA. The obtained rbcL sequences were compared to databases in GenBank and Barcode of Life Database (BOLD) to identify the plant species.