Labnotes

None

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
  1. Gibson assembly (Kit)
  • 20ul system
  • 10ul 2x clone mix
  • 0.5ul plasmid template
  • ddH2O up to 20ul
  • Pipet to mix
  • PCR 50°C 50min
  1. 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
  1. 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.
  1. 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.