Experiments

Table of Contents

1. Recovering Plasmid DNA

Materials:

  1. Overnight culture of bacteria transformed with plasmid
  2. Plasmid Mini Kit
  3. Sterilized distilled water

Equipment:

  1. Eppendorf tube
  2. Desktop microcentrifuge
  3. Desktop vortexer
  4. thermostat water bath

Procedures:

  1. Grow an overnight culture of bacteria.
  2. Centrifuge the 1~5mL culture to pellet the bacteria before proceeding with DNA preparation.
  3. Remove the supernatant and resuspend the bacteria in buffer.
  4. Add a denaturing solution from the Plasmid Mini Kit to the resuspended bacteria.
  5. Add a renaturing solution from the Plasmid Mini Kit to the denatured bacteria.
  6. Pellet the proteins and genomic DNA by centrifugation and remove the plasmid-containing supernatant.
  7. Add ethanol to precipitate the plasmid DNA.
  8. Centrifuge at 12000rpm for 10 min to pellet the DNA.
  9. Wash the pellet with 70% ethanol to remove excess salt.
  10. Resuspend the DNA pellet Sterilized distilled water.

2. Diagnostic Endonuclease Digest

Materials:

  1. Plasmid DNA
  2. Restriction enzyme
  3. Restriction digest buffer
  4. Forward Primer
  5. Reverse Primer
  6. 10X PrimeSTAR GXL Buffer with MgCl2
  7. dNTP mix
  8. PrimeSTAR GXL DNA Polymerase

Equipment:

  1. Thin-walled PCR tube
  2. Ice box
  3. PCR Machine

Procedures:

  1. In a 1.5mL tube combine the following:
    1. 1μg of plasmid DNA sample
    2. 1μL of each restriction enzyme
    3. 3μL restriction digest buffer
    4. H2O up to total volume of 30μL
  2. Mix gently by pipetting.
  3. Incubate tube at 37°C for 1hour.
  4. Place thin-walled PCR tubes on ice. Set up a 50μL reaction:
    1. 1μL template DNA
    2. 5μL 10X PrimeSTAR GXL Buffer with MgCl2
    3. 4μL dNTP mix
    4. 2μL of 10μM Forward Primer
    5. 2μL of 10μM Reverse Primer
    6. 1μL PrimeSTAR GXL DNA Polymerase
    7. H2O up to total volume of 50μL
  5. Place reaction tubes in PCR machine. Set the standard time and temperature for each step in a PCR cycle:
    1. Initial Denaturation for 2min at 98°C.
    2. Denature for 10s at 98°C.
    3. Anneal primers for 30s at 55°C.
    4. Extend DNA for 1min at 72°C.
    5. Repeat b-d steps for 20-30 cycles.
    6. Extension for 5 min at 72°C.
  6. The products were stored at 4 °C.

3. Acquisition of Target Gene Fragment

Materials:

  1. 10X PrimeSTAR GXL Buffer with MgCl2
  2. template DNA
  3. Forward Primer
  4. Reverse Primer
  5. dNTP Mixture
  6. PrimeSTAR GXL DNA Polymerase
  7. Sterilized distilled water

Equipment:

  1. Eppendorf tube
  2. Desktop microcentrifuge
  3. Desktop vortexer
  4. Ice box
  5. PCR instrument

Procedures:

  1. Place thin-wall PCR tubes on ice. Set up a 50μL reaction:
    1. 1μL template DNA
    2. 5μL 10X PrimeSTAR GXL Buffer
    3. 4μL dNTP mix
    4. 2μL of 10μM Forward Primer
    5. 2μL of 10μM Reverse Primer
    6. 1μL PrimeSTAR GXL DNA Polymerase
    7. H2O to a total of 50μL
  2. Place reaction tubes in PCR machine. Set the standard time and temperature for each step in a PCR cycle:
    1. Initial Denaturation for 2min at 98°C.
    2. Denature for 10 seconds at 98°C.
    3. Anneal primers for 15s at 55°C below.
    4. Extend DNA for 1min at 72°C.
    5. Repeat b-d steps for 30 cycles
    6. Extension for 5 min at 72°C
    7. The products were stored at 4°C.

4. Construction of Recombinant Plasmids

Materials:

  1. Linearized vector plasmid DNA
  2. Target gene segment
  3. DNA ligase and buffer
  4. Competent state Escherichia coli DH5α
  5. Luria-Bertani media

Equipment:

  1. Eppendorf tube
  2. Shaking incubator at 37°C
  3. Stationary incubator at 37°C
  4. Water bath at 42°C
  5. Ice box
  6. Microcentrifuge tubes
  7. Sterile spreading device

Procedures:

  1. Combine the following in an Eppendorf tube:
    1. 2.5μL Plasmid Vector DNA
    2. 2μL Insert DNA (For a control group, add H2O instead of insert DNA)
    3. 2μL Ligase Buffer
    4. 1μL DNA Ligase
    5. H2O to a total of 20μL
  2. Incubate at 37°C for 30min before cooling in an ice water bath for 5min.
  3. Retrieve competent cells from -80°C refrigerator and defrost on ice for approximately 10minutes.
  4. Retrieve the agar plates containing the appropriate antibiotic from storage at 4°C, and let them warm up to room temperature.
  5. Mix 10μL of plasmid DNA into 100μL of competent cells in a microcentrifuge tube. Gently mix the components. For a control group, add 10μL of water to verify that the LB agar’s antibiotic functions properly.
  6. Incubate the competent cell/DNA mixture on ice for approximately 30minutes.
  7. Heat shocks each transformation tube by placing the bottom half of the tube into a 42°C water bath, leaving it for approximately 45s.
  8. Put the tubes back on ice for 2min.
  9. Add 300μL LB media (without antibiotic) to the bacteria, and grow in 37°C shaking incubator for 30 min.
  10. Plate an appropriate amount of the transformation onto the LB agar plate containing the corresponding antibiotic. Incubate plates at 37°C overnight.

5. Agarose Gel Electrophoresis

Materials:

  1. DNA samples
  2. Gel loading dye
  3. 10X TAE bufferl
  4. Agarose
  5. 4S green plus

Equipment:

  1. Well combs
  2. Gel box
  3. Microwave
  4. Electrophoresis chamber
  5. Voltage source
  6. UV light source
  7. Casting tray

Procedures:

  1. Mix 0.2g of agarose powder and 1μL of 4S green plus with 20mL 1XTAE in a microwavable flask.
  2. Microwave for 1-3 min until the agarose is completely dissolved.
  3. Let agarose solution cool down to about 50°C in about 5 min.
  4. Pour the agarose into a gel tray with the well comb in place.
  5. Place the gel at room temperature for 20-30 min, until it has completely solidified.
  6. Place the gel into the gel box of electrophoresis unit. Fill gel box with 1xTAE until the gel is covered.
  7. Carefully load a molecular weight ladder.
  8. Add loading buffer to each DNA sample. Carefully load samples into the additional wells of the gel.
  9. Run the gel at 120 V for approximately 15 min.
  10. Turn off power, disconnect the electrodes, and remove the gel from the gel box.
  11. Visualize DNA fragments using UV light source.

6. Recombinant Protein Expression

Materials:

  1. Recombinant Escherichia coli BL21
  2. Antibiotic
  3. 1M Isopropyl-β-D-thiogalactopyranoside（IPTG）
  4. Luria-Bertani media

Equipment:

  1. 50mL shaking flask
  2. Shaking incubator at 37°C
  3. Shaking incubator at 18°C
  4. Stationary incubator at 37°C

Procedures:

  1. Pick a single colony from the plate and add it to 10mL of LB medium, incubate at 37°C overnight with shaking.
  2. 5mL of the above bacteria solution was added to 1L1L of LB medium, incubated at 37°C with shaking until the OD600 was between 0.40~0.45, and left to stand at room temperature for 45min.
  3. incubate at 18°C for 45min.
  4. Add 200uL of 1M IPTG solution and incubate at 18°C for 16~18h.
  5. Centrifuge at 4°C and discard the supernatant to collect the bacteria..

7. Extraction and Verification of Protein

7.1. Extraction of Protein

Materials:

  1. Recombinant Escherichia coli BL21
  2. Lysis buffer
  3. Elution buffer
  4. SDS buffer

Equipment:

  1. Eppendorf tube
  2. 50mL Centrifuge tube
  3. HisCap IDA 6FF
  4. Prepacked Desalting Column
  5. Amicon® Ultra
  6. Desktop centrifuge
  7. Metal bath pot
  8. Centrifugal machine
  9. Ultrasonic cell breaker
  10. ÄKTA pure

Procedures:

  1. Add 40mL Lysis buffer to the cell, resuspend the cells, and ultrasonically break the cells on ice for 1h.
  2. After centrifugation at 11000rpm for 40min at 4°C, the supernatant was collected and passed through a 0.22um filter membrane.
  3. Separate the protein samples using ÄKTA pure and combine the target proteins according to the UV absorption peaks.
  4. Imidazole was removed from the solution by Prepacked Desalting Column.
  5. Concentrate the protein using Amicon® Ultra and store at -80°C in a refrigerator.

7.2. Verification of Protein

Materials:

  1. SDS-PAGE gel
  2. SDS-PAGE running buffer
  3. Loading buffer
  4. Protein Ladder
  5. Coomassie brilliant blue G-250
  6. Coomassie Blue Staining Destaining Solution

Equipment:

  1. SDS-PAGE electrophoresis tank
  2. SDS-PAGE transfer tank
  3. Microwave
  4. Imager System

Procedures:

  1. The SDS-PAGE gel was placed in the electrophoresis tank. Connect electrode and load 20µL of each sample into the gel.
  2. Run the gel at 100 V for approximately 20 minutes, until the dye has migrated into the running gel.
  3. Increase the voltage to 150 V and continue for approximately 30 minutes, until the dye front has reached the bottom of the gel.
  4. Place the gel into a small tray. Add Coomassie brilliant blue G-250 and heat for 1 min.
  5. Pour off the Coomassie brilliant blue G-250 and add dehydration solution for rinsing.
  6. Put the gel into the imager and select the appropriate exposure time, and save the image.

8. Functional Verification of protein

Materials:

  1. Protein
  2. modified nucleotides
  3. Oligonucleotides
  4. PBS
  5. CoCl2 solution
  6. TBE buffer
  7. SYBR™ Gold
  8. Sterilized distilled water

Equipment:

  1. Thin-walled PCR tube
  2. Ice box
  3. PCR Machine

Procedures:

  1. Catalytic activity of protein was determined in 30µL reaction mixtures.
  2. The reaction system comprised 1uM Oligonucleotides, 0.1 mM dNTPs, 0.25mM CoCl2 and PBS.
  3. The catalytic reaction was started by adding 0.5 mg/mL purified protein.
  4. After incubation at 30 °C for 20 min, the reaction was stopped by heating at 95°C.
  5. The products of the reaction were analyzed by gel electrophoresis on a 20% acrylamide gel and 8M urea.
  6. Subsequently, the PAGE gel was stained with SYBR™ Gold nucleicacid gel dye for 20 minutes and scanned with a fully automatic fluorescence image analysis system.
  7. The result was processed with Gel Image System analysis software.