Cloning

Gene Fragment PCR

Primer Dilution 

  1. in order to make 10 µM primer
  2. 20 µL primer with 180 µL dH2O

PCR

  1. PCR master mix was prepared by including the following components (per reaction): Master Mix (2X Premix), 8 µl; Forward Primer (10 µM), 1 µl; Reverse Primer (10 µM), 1 µl; dH2O, 6 µl. PCR master mix aliquots (16 µl) were dispensed into each PCR tube. (should make more than you need) (should make one without the sample)
  2. A sterile micropipette tip was used to transfer a few cells from each colony to a corresponding PCR tube, where cells were resuspended in PCR master mix.
  3. The cycling conditions were: 94°C, 5 min; followed by 35 cycles of 94°C, 30 sec; 55 (Tm)°C, 30 sec; and 72°C, 45 sec, 72°C, 5 min; 4°C, hold. After PCR was complete, 10µl of each reaction was subjected to electrophoresis on a 0.8% agarose gel.

PCR cleanup

  1. Insert the binding column into a provided collection tube.
  2. Add 0.5 mL of the column preparation solution to each miniprep column.
  3. Centrifuge at 12,000 x g for 1 minute.
  4. After 1 minute, dispose of the eluate.
  5. Transfer the PCR reaction mix into each tube.
  6. Combine 5 parts of binding solution with 1 part of the PCR reaction and thoroughly mix.
  7. Transfer the solution into the binding column.
  8. Centrifuge at maximum speed for 1 minute.
  9. After 1 minute, discard the eluate, but keep the collection tube.
  10. Add 0.5 mL of diluted wash solution to the column.
  11. Centrifuge at maximum speed for 1 minute.
  12. After 1 minute, dispose of the eluate, and return the column to the collection tube.
  13. Centrifuge at maximum speed for 2 minutes without adding additional wash solution.
  14. After 2 minutes, discard any remaining eluate and the collection tube. Transfer the column to a new 2 mL collection tube.
  15. Add 50 µL of elution solution to the center of each column.
  16. Centrifuge at maximum speed for 1 minute.
  17. After 1 minute, discard the column.
  18. The eluate now contains the PCR amplification products.

Plasmid Extraction

  1. To collect the bacterial culture, centrifuge 1.5 ml in a microfuge tube at 10,000 rpm for 2 minutes.
  2. Remove the liquid (supernatant) and introduce 200 µl of resuspension buffer. To fully mix the pellet, you may need to vortex.
  3. Incorporate 250 µl of lysis buffer and gently invert the tube about 10 times to ensure thorough mixing. The solution should become clear and viscous.
  4. Add 350 µl of neutralization buffer and invert the tube approximately 10 times or until a precipitate forms. This precipitate is a combination of protein and chromosomal DNA.
  5. Centrifuge the tube at 10,000 rpm for 10 minutes. Transfer the resulting supernatant to a microfuge tube, and add 0.7 isopropanol. Incubate at -20°C for 15 minutes.
  6. Transfer the mixture to a spin column.
  7. Centrifuge the spin column for 1 minute at 7,000 rpm. Dispose of the liquid that flows through.
  8. Add 400 µl of wash buffer to the column and centrifuge for 1 minute at 7,000 rpm. Discard the liquid that flows through. Repeat this step.
  9. Centrifuge for an additional 2 minutes at 10,000 rpm to remove any remaining wash buffer.
  10. Transfer the column to a clean microfuge tube. Add 50 µl of elution buffer and centrifuge for 1 minute at 10,000 rpm.

Digeston

combine the following components in a clean tube in the specified order:

  1. 1 µL of DNA (concentration: 1 µg/µL)
  2. 2 µL of 10x buffer
  3. 1 µL of each restriction enzyme
  4. 15 µL of sterile water

   Next, incubate the reaction at the digestion temperature, which is typically 37 °C, for a duration of 1 hour. 
   To halt the digestion process,: Heat inactivates by placing the reaction tube at 65°C for 15 minutes. Alternatively, you can stop the digestion by adding EDTA to achieve a final concentration of 10 mM. 
   Once you have completed these steps, the digested DNA will be ready for research applications. 

Gel extraction

  1. Carefully cut out the gel slice containing the DNA fragment using a clean scalpel or razor blade. Trim as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and record its weight.
  2. Heat the elution buffer (EB) to 65 degrees Celsius using a water bath.
  3. Add a volume of Solution Buffer to the gel slice in a 3:1 ratio (volume to weight). For example, add 300 µL of Binding Buffer for every 100 mg of agarose gel. Incubate the gel mixture at 60 degrees Celsius for at least 5 minutes, or until the gel slice is completely dissolved. Invert the tube every few minutes to aid the melting process. Observe the color of the solution; a yellow color indicates an optimal pH for DNA binding. If the solution appears orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix until the color turns yellow.
  4. Transfer the solution to a fresh adsorption column. Centrifuge at 13,000 rpm for 1 minute, and then pour off the liquid from the collection tube. If necessary, repeat this step for critical samples.
  5. Before centrifugation, add 600 µL of washing buffer (WB). Centrifuge at 13,000 rpm for 1 minute, and then pour off the liquid into a beaker.
  6. Centrifuge at 13,000 rpm for 10 minutes to remove residual ethanol from the pellet.
  7. Place the column into a fresh EP (Eppendorf) tube. If required, air-dry the pellet for 10-15 minutes to ensure there is no residual ethanol left in the purified DNA solution. Residual ethanol in the DNA sample may inhibit downstream enzymatic reactions.
  8. Add 30-50 µL of elution buffer (EB) to elute the DNA.
  9. Use 5 µL of the eluted sample for identification through electrophoresis.

Ligation

  1. Combine the following components in a PCR or Eppendorf tube:
    Vector DNA
    Insert DNA 
    Ligase Buffer (1 μL for every 10 μL reaction for 10X buffer, and 2 μL for every 10 μL reaction for 5X buffer) 0.5-1 μL of T4 DNA Ligase Add H2O to reach a total volume of 10 μL 
    Note: Because the ligase buffer contains ATP, which degrades upon freeze/thaw cycles, it's advisable to use a fresh tube. Thaw it once, and then aliquot individual tubes of 5, 10, or 20 μL for storage at -20°C. In the future, when setting up ligations, use a newly thawed tube that has only been thawed once before. 
    Note: Always include control experiments. 
    Note: Experiment with different vector-to-insert ratios to optimize the ligation reaction. 
  2. Incubate the mixture at room temperature for 2 hours or at 16°C overnight, following the manufacturer's instructions.

E. coli Transformation

  1. Pipette 10µL of dH2O (distilled water) into the well. The resuspension will be red-ish, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA. 
  2. Transfer the required number of tubes from -70° C freezer to wet ice. Include an extra tube for control DNA, if desired
  3. Allow the cells to thaw for 5 minutes. Gently tap the tubes multiple times to obtain uniform suspension 
    a. For control: Add 1 µL (10 ng) pUC19 control DNA to one tube. Mix by gentle tapping and place on ice 
    b. Add 1 ng to 50 ng of purified plasmid DNA directly to cells in the rest of the tubes. Mix by gentle tapping and place on ice
  4. Transform 1µL of the resuspended DNA into 25 µL of your desired competent cells (DH5α)
  5. Incubate the cells on ice for 10 minutes
  6. Transfer the cells to 42° C water bath for exactly 60 seconds
  7. Transfer the cells to ice for 2 minutes
  8. Add 1mL LB medium（without amp) to each tube. Transfer the cells to sterile culture tubes and loosen the caps to facilitate aeration of the cultures
  9. Incubates the cells on shaker incubator (50 rpm) at 37° C for 1 hour
  10. centrifuge each tube at 8000 for 1 minute and draw some supernatant
  11. Pipette 10-100 µL of each transformed cell suspension onto LB agar plates with selection antibiotic and spread it using sterile spreader
  12. Incubate plates at 37° C overnight
  13. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
  14. Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the minipreps DNA to run QC tests, such as restriction digests and sequencing.

   Next, incubate the reaction at the digestion temperature, which is typically 37 °C, for a duration of 1 hour. 
   To halt the digestion process,: Heat inactivates by placing the reaction tube at 65°C for 15 minutes. Alternatively, you can stop the digestion by adding EDTA to achieve a final concentration of 10 mM. 
   Once you have completed these steps, the digested DNA will be ready for research applications. 

Yeast Transformation

Preparing Electrocompetent Cells: 

  1. Cultivate a 50 ml cell culture of Kluyveromyces lactis until it reaches a density of 5 x 10^7 cells/ml.
  2. Harvest the cells through centrifugation (5 minutes at 4,000 x g).
  3. Resuspend the harvested cells in 5 ml of a washing solution and let them incubate for 5 minutes.
  4. Perform two additional washes using electroporation solution and resuspend the cells in 0.25 ml of the same solution. Allow them to incubate for 1 hour on ice.

Electroporation of Cells:

  1. Combine 5 µl of DNA (1 µg/µl) with 100 µl of electrocompetent cells. Mix gently by pipetting several times. Transfer this mixture into a pre-chilled cuvette.
  2. Wipe off any moisture from the cuvette and insert it into the electroporation device.
  3. Configure the electroporation settings as follows: 
    - Mode: Prokaryotes 
    - Voltage: 2,500 V 
    - Time constant (τ): 5 ms 
    - Conduct the electroporation.

Post-Electroporation Steps:

  1. After electroporation, add 1 ml of YPD medium with 1 M sorbitol and incubate for 1 hour at 30 °C.
  2. Centrifuge the cells for 30 seconds and then resuspend them in 0.4 ml of sterile water.
  3. Plate the cells in aliquots on selective plates for further experimentation. 

Bacillus Transformation

Preparing Electrocompetent Cells: 

  1. Cultivate a 50 ml cell culture of Kluyveromyces lactis until it reaches a density of 5 x 10^7 cells/ml.
  2. Harvest the cells through centrifugation (5 minutes at 4,000 x g).
  3. Resuspend the harvested cells in 5 ml of a washing solution and let them incubate for 5 minutes.
  4. Perform two additional washes using electroporation solution and resuspend the cells in 0.25 ml of the same solution. Allow them to incubate for 1 hour on ice.

Electroporation of Cells:

Stationary phase culture of B. subtilis (18 to 24 hrs of incubation time) in 10 mL LB Miller medium:

  • Purified plasmid (1 μL)
  • SOC medium (0.9 mL)
  • 20% xylose solution (100 μL)
  • LB agar plates with appropriate antibiotics

Procedure

  1. Start by obtaining a stationary phase culture of B. subtilis. Incubate the culture at 30°C and 225 rpm for 18 to 24 hours after inoculating glycerol stock culture in 10 mL LB Miller medium.
  2. Aliquot 1.6 mL of the stationary phase culture into a microcentrifuge tube
  3. Centrifuge the contents of the microcentrifuge tube at 3300 x g for 2 minutes to pellet the cells. 
  4. Carefully discard the supernatant, leaving the cell pellet intact.
  5. Add 1 μL of purified plasmid directly to the cell pellet. Gently flick the tube to mix the plasmid with the cells, ensuring thorough and gentle resuspension.
  6. To the cell suspension, add 0.9 mL of SOC medium and 100 μL of a 20% xylose solution. Vortex the cell suspension gently to mix all components evenly. Incubate the cells at 30°C for 2 hours.
  7. After 2 hours of incubation, aliquot 200 μL of the cell suspension onto LB agar plates that contain the appropriate antibiotics for plasmid selection.
  8. Incubate the agar plates at 30°C overnight to allow the transformed cells, which have taken up the plasmid, to form colonies.

Colony PCR

Gel Preparation

  1. 0.8% - 2% TAE gel at 50 °C
  2. add 2 μl DNA stain into a centrifuge tube, and add TAE gel to 40 mL

Electrophoresis

  1. Pour gel into the tray and wait 30 min (until become jelly-like)
  2. clear (wipe) the tray
  3. Peel off the lid. Leave the gel in the tray.
  4. Press the tray directly onto the chamber platform. Align the wells so the NA samples will run straight.
  5. Pour buffer to a depth of 5 mm OVER the flange of the tray.
  6. Load the DNA sample (10 μl volume).
  7. Electrophorese the gels at 100 V for 20 min.
  8. Remove the gel from the tray to photograph/document and/or destine.

Glycerol Stock

  1. Add 30%glycerol into the desired polypropylene tube with ratio mixture:glycerol=1:1, making it 15% glycerol (maybe 500µL:500µL)
  2. Pipette up and down in the culture tube
  3. Store at -80°C

Inoculation for culture

Liquid culture:

  1. Prepare for 3-4 mL of LB or YPD medium

Cell Culture

Liquid culture:

  1. Prepare for 3-4 mL of LB or YPD medium
  2. Use tip to touch the culture you select and mix it with the liquid

Plate culture:

  1. Prepare for plate with appropriate antibiotics
  2. Use tip to burn by fire and touch the culture selected and touch the plate

Phenotyping

HPLC:

  1. Obtain a sample of the target material.
  2. Dilute the sample with water to achieve a specific concentration.
  3. Create a calibration curve using samples with various concentrations.
  4. Introduce the target material into bacteria or yeast cells that have already undergone cell lysis using different protocols.
  5. Calculate the productivity of the bacteria or yeast based on the experimental data and calibration curve.