Preparation of Reagents

Table 1. materials of SOB medium per liter:

ComponentAmount
Yeast Extract5 g
Tryptone20 g
KCl0.186 g
MgCl₂3.808 g
NaCl0.584 g

Table 2. materials of LB medium (liquid/agar) per liter:

ComponentAmount
Yeast Extract5 g
Tryptone10 g
NaCl10 g
Agar (for solid medium)20 g

Preparation of LB/SOB medium Steps:

  1. Take an empty reagent bottle and rinse it with milliQ water
  2. Use electronic balance, weighting boat and a spatula to add the required amounts of materials into the reagent bottle
  3. Add water to the volume needed
  4. Close the lid of the reagent bottle and autoclave it at 121°C for 15-20 minutes

PCR Amplification

1. Transfer contents into a 0.2 mL PCR tube according to the following recipe:

Component25µL Reaction50µL Reaction (for gel extraction)
Q5 High-Fidelity 2× Master Mix12.5 µL25 µL
Forward Primer (10 µM)1.25 µL2.5 µL
Reverse Primer (10 µM)1.25 µL2.5 µL
Template DNA20 ng20 ng
Nuclease-Free Waterto 25 µLto 50 µL

2. Set the PCR Thermocycling Conditions:

StepTemperatureTime
Initial Denaturation98°C30 sec
35 Cycles98°C10 sec
35 Cycles

68

70.5°C

30 seconds (for MNEIs and TAS1R3)

30 seconds (for TAS1R2)

35 Cycles72°C

10 seconds (for MNEIs)

25 seconds (for TAS1R2/3)

Final Extension72°C2 min
Hold4°C∞

DNA purification

DNA Fragment purification

  1. Add a 3X volume of Buffer DC to the PCR or other enzymatic reactions.
  2. If the required volume of Buffer DC is less than 100 μL, add 100 μL of Buffer DC. Vortex briefly to mix the sample.Place a Spin Column into a Collection Tube.
  3. Pipette the sample from Step 1 into the Spin Column. Centrifuge at 12,000 rpm for 1 minute. Dis the flow-through.
  4. Note: For improvement the recovery of DNA, transfer the flow-through to Spin Column and centrifuge again.Add 700 μL Buffer WB to Spin Column. Centrifuge at 12,000 rpm for 30 seconds. Dis the flow-through.
  5. Note: Make sure that the volume of 100% ethanol indicated on the bottle label has been added to the Buffer WB.Repeat Step 4.
  6. Place the Spin Column back into the Collection Tube. Centrifuge at 12,000 rpm for 1 minute.
  7. Place the Spin Column into a new clean 1.5 ml tube. Add 25 −30 μL Elution Buffer or sterilized water to the centre of the membrane.
  8. Let it stand for 1 minute at room temperature.

  9. Note

    • Pre-heat the Elution Buffer or sterilized water to 60℃ can increase the elution efficiency.
    • For highest recovery, Incubate for 1 and a half hour / as long as possible at 37℃.
  10. Centrifuge at 12,000 rpm for 1 minute at room temperature to elute DNA.

Plasmid purification

  1. Growth of bacterial cultures. Pick a single colony from a freshly streaked selective plate into 1 - 4 mL of liquid culture containing the appropriate selective antibiotic. Incubate at 37℃ overnight with vigorous shaking. (Growth for 12 - 16 hours and do not exceed 16 hours, otherwise the bacterial cells will be hard to lyse and the yield of plasmid DNA will be reduced.)
  2. Note: The culture volume should not be excessive because excessive bacteria will decrease lysis efficiency and result in poor purity of plasmid DNA.Use 1 - 4 mL of the E. coli culture. Centrifuge at 12,000 rpm for 2 minutes to harvest the cell. Dis the supernatant.
  3. Add 250 μL Solution l (containing RNase A). Resuspend the bacterial cell pellet completely by pipetting up and down.
  4. Note: Be sure that the bacteria are completely resuspended, and no cell clumps remain before addition of Solution ll.Add 250 μL Solution ll and mix gently by inverting the tube 5 - 6 times to completely lysis the cell until the solution becomes viscous and slightly clear.
  5. Note: Do not allow the lysis reaction to proceed more than 5 minutes.Add 350 μL of 4℃ precooling Solution lll and mix immediately and thoroughly by inverting the tube 5 - 6 times until a compact white pellet has been formed. Incubate at room temperature for 2 minutes.
  6. Centrifuge at 13,200 rpm at room temperature for 10 minutes.
  7. Note: Centrifuging at 4℃ is not recommended for precipitation.Apply the supernatant from Step 6 into a new clean 1.5 mL tube by precise pipetting. Centrifuge at 13,200 rpm at room temperature for 10 minutes.
  8. Note: The white precipitate will clog the spin column, reducing the yield.Place a Spin Column in a Collection Tube.
  9. Apply the supernatant from Step 6 onto the Spin Column by pipetting. Centrifuge at 12,000 rpm for 1 minute. Dis the flow-through with the aid of pipette.
  10. Pipette 500 μL of Buffer WA onto the Spin Column. Centrifuge at 12,000 rpm for 1 minute. Dis the flow-through with the aid of pipette.
  11. Pipette 700 μL of Buffer WB onto the Spin Column. Centrifuge at 12,000 rpm for 1 minute. Dis the flow-through with the aid of pipette.
  12. Note: Make sure that the amount of 100% ethanol indicated on the bottle label has been added to Buffer WB.Repeat Step 11.
  13. Place the Spin Column back into new 1.5 mL tube without a lid. Centrifuge at 13,200 rpm for 5 minutes. Dis the flow-through with the aid of pipette.
  14. Place the Spin Column back into 1.5 mL tube without a lid. Centrifuge at 13,200 rpm for 3 minutes. Dis the flow-through with the aid of pipette.
  15. Note: Residual ethanol from Buffer WB may inhibit subsequent enzymatic reaction.Place the Spin Column in a new clean 1.5 mL tube. Add 50 μL Elution Buffer or sterile purified water to the center of the Spin Column membrane. Incubate for 1 and a half hour at 37℃. Note: Pre-heat Ultra-purified wter to 60℃
  16. Centrifuge at 13,200 rpm for 1 minute to elute DNA.

Agarose Gel Electrophoresis

  1. Measure 0.2g of agarose.
  2. Mix agarose powder with 20 mL 1xTAE in a microwavable flask.
  3. Microwave until the agarose is completely dissolved.
  4. Dilute the GelGreen® 10,000X stock reagent into the molten agarose gel solution at 1:10,000 and mix thoroughly. GelGreen® can be added while the solution is still hot.
  5. Cast the gel and allow it to solidify. Load samples and run the gels (90V, 30 mins).
  6. Image the stained gel with a 254 nm UV transilluminator or 490nm to 500nm visible light imaging system with a long path green filter.

Preparation of NiCo21/DH5-ɑ Competent Cell

Prepare Inoue transformation buffer (chilled to 0°C before use).

  1. Prepare 0.5 M PIPES (pH 6.7) (piperazine-1,2-bis[2-ethanesulfonic acid]) by dissolving 15.1 g of PIPES in 80 ml of pure H2O (Milli-Q, or equivalent).
  2. Adjust the pH of the solution to 6.7 with 5 M KOH, and then add pure H2O to bring the final volume to 100 ml. Sterilize the solution by filtration through a disposable pre-rinsed Nalgene filter (0.45-µm pore size). Divide into aliquots and store frozen at -20°C.

Preparation of Inoue transformation buffer

  1. Dissolving all of the solutes listed below in 800 ml of pure H2O and then add 20 ml of 0.5 M PIPES (pH 6.7). Adjust the volume of the Inoue transformation buffer to 1 liter with pure H2O.

    2. 2. Reagent Amount per liter Final concentration:

    ReagentAmount per literFinal concentration
    MnCl2•4H2O10.88g55 mM
    CaCl2•2H2O2.20g15mM
    KCl 18.65g250 mM
    PIPES (0.5 M, pH 6.7)20mL10 mM
  2. Sterilize Inoue transformation buffer by filtration through a prerinsed 0.45-µm Nalgene filter. Divide into aliquots and store at -20°C.
  3. Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated for 16-20 hours at 37°C. Transfer the colony into 25 mL of SOB medium (LB may be used instead) in a 250-mL flask. Incubate the culture for 6-8 hours at 37°C with vigorous shaking (250-300 rpm).
  4. At about 6 o'clock in the evening, use this starter culture to inoculate three 1-liter flasks, each containing 250 mL of SOB. The first flask receives 10 mL of starter culture, the second receives 4 mL, and the third receives 2 mL. Incubate all three flasks overnight at 18-22°C with moderate shaking.
  5. The following morning, read the OD600 of all three cultures.Continue to monitor the OD every 45 minutes.
  6. When the OD600 of one of the cultures reaches 0.55, transfer the culture vessel to an ice-water bath for 10 minutes. Dis the two other cultures.
  7. Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for10 minutes at 4°C.
  8. Pour off the medium and store the open centrifuge bottle on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to walls of the centrifuge bottle or trapped in its neck.
  9. Resuspend the cells gently in 80 mL of ice-cold Inoue transformation buffer.
  10. Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for 10 minutes at 4°C.
  11. Pour off the medium and store the open centrifuge tube on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to the walls of the centrifuge tube or trapped in its neck.
  12. Resuspend the cells gently in 20 mL of ice-cold Inoue transformation buffer.
  13. Add 1.5 mL of DMSO. Mix the bacterial suspension by swirling and then store it in ice for 10 minutes.
  14. Working quickly, dispense aliquots of the suspensions into chilled, sterile microcentrifuge tubes. Immediately snap-freeze the competent cells by immersing the tightly closed tubes in a bath of liquid nitrogen. Store the tubes at -70°C until needed.
  15. When needed, remove a tube of competent cells from the -70°Cfreezer. Thaw the cells by holding the tube in the palm of the hand. Just as the cells thaw, transfer the tube to an ice bath. Store the cells on ice for 10 minutes.
  16. Use a chilled, sterile pipette tip to transfer the competent cells to chilled, sterile 17 x 100-mm polypropylene tubes. Store the cells on ice. Include all of the appropriate positive and negative controls.
  17. Add the transforming DNA (up to 25 ng per 50 µl of competent cells) in a volume not exceeding 5% of that of the competent cells. Swirl the tubes gently several times to mix their contents. Set up at least two control tubes for each transformation experiment, including a tube of competent bacteria that receives a known amount of a standard preparation of superhelical plasmid DNA and a tube of cells that receives no plasmid DNA at all. Store the tubes on ice for 30 minutes.
  18. Transfer the tubes to a rack placed in a preheated 42°C circulating water bath. Store the tubes in the rack for exactly 90 seconds. Do not shake the tubes.
  19. Rapidly transfer the tubes to an ice bath. Allow the cells to cool for 1-2 minutes.
  20. Add 800 µl of SOC medium to each tube. Warm the cultures to 37°C in a waterbath, and then transfer the tubes to a shaking incubator set at 37°C. Incubate the cultures for 45 minutes to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid.
  21. Transfer the appropriate volume (up to 200 µl per 90-mm plate) of transformed competent cells onto agar SOB medium containing 20 mM MgSO4 and the appropriate antibiotic.
  22. Store the plates at room temperature until the liquid has been absorbed.
  23. Invert the plates and incubate them at 37°C. Transformed colonies should appear in 12-16 hours.

Transformation

Bacteria Transformation

  1. Thaw competent E. coli cells on ice.
  2. Add 1–4 ng plasmid DNA and incubate 30 minutes.
  3. Incubate for 30 minutes on ice.
  4. Heat shock at 42℃ for 45 seconds.
  5. Incubate for 30 minutes on ice.
  6. Add LB medium so total volume is 1 mL.
  7. Recover cells for 90 minutes in a shaker at 37°C at 270 r.m.p.
  8. Centrifuge cells at 3000xg for 3 minutes.
  9. Remove all liquid medium from the tube.
  10. Resuspend in 100 ul of LB medium.
  11. Spread plates using glass plates.
  12. Add 6 beads on either side of each plate.
  13. Flicker tube until cells are uniformly distributed.
  14. Gently pipette a dilution of outgrowth onto the centre of the pre-warmed plate.
  15. Vigorously move the plates forward and backwards. Make two complete 360° rotations, with stops every 90 degrees.
  16. Allow plates to sit for 2 minutes.
  17. Pour off the beads into a beaker.
  18. Incubate plates at 37°C.

Restriction Enzyme Digestion

  1. Transfer contents into a 0.2mL PCR tube according to the following recipe:
    2. Component50µL Reaction
    DNA1 µL
    10X CutSmart Buffer5µL (1X)
    NcoI-HF1.0µL (20 units)
    XhoI1.0µL (20 units)
    Nuclease-free Waterto 50 µL
  2. Heat inactivation (65°C for EcoRI-HF® & 80°C for NheI-HF®)

Ligation

  1. Transfer contents into a 1.5 mL microcentrifuge tube according to the following recipe on ice:
    2. Component20µL reaction
    T4 DNA Ligase Buffer (10×)2 µL
    Vector DNAvaries
    Insert DNAvaries
    T4 DNA Ligase1 µL
    WaterTop up to 20 µL
  2. Use NEBioCalculator to calculate molar ratios.
  3. The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.
  4. Gently mix the reaction by pipetting up and down and microfuge briefly.
  5. Incubate at 16°C overnight