Team:Jilin China/Experiments

PROTOCOLS


Protocols

  • Protocols

    1. Annealing Oligos

    Experimental procedure：

    Reagent volume
    Oligo1 (50μM) 9μl
    Oligo2 (50μM) 9μl
    T4 DNA Ligase Buffer 2μl
    Total 20μl

    1. Label the 0.2 ml eppendorf tubes.

    2. Add all the reagents to the labeled eppendorf tube in ice bath.

    3. Blend the reagents in the 0.2 ml eppendorf tube by short centrifugation.

    4. Place the 0.2 ml eppendorf tubes into PCR instrument and run the annealing program.

    Instrument flow：

    step1：95℃——3min

    step2：94℃——1min

    step3：93℃——1min-1.0℃/cycle

    step4：56 times to step3

    step5：37℃——1min

    step6：4℃——∞

    step7：END

    2. Phosphorylation

    Experimental procedure：

    Reagent volume
    Oligo Annealing Products 4µl
    PNK 0.4µl
    T4 DNA Ligase Buffer 1.0µl
    ddH2O 4.6µl
    Total 10μl

    MIX=PNK+T4 DNA Ligase Buffer+ddH2O

    1. Label the 1.5 ml eppendorf tubes and the 0.2ml eppendorf tubes.

    2. First configure enough MIX according to the amount of experiment requirement.

    3. Add all the reagents to a 0.2 ml eppendorf tube in ice bath.

    4. Blend the reagents in the 0.2 ml eppendorf tube by short centrifugation.

    5. Place the 0.2 ml eppendorf tubes into PCR instrument and run the phosphorylation program.

    Instrument Flow:

    Step1: 37℃——60min

    Step2: 4℃——∞

    Step3: END

    3. Golden Gate Assembly

    Experimental Procedure:

    Reagent volume
    Phosphorylation Products 1µl
    T4 DNA Ligase 0.2µl
    T4 DNA Ligase Buffer 0.5µl
    Type IIS Restriction Endonuclease 0.2µl
    Backbone Plasmid 0.2µl (~200ng)
    ddH2O 2.9µl
    Total 5μl

    MIX=T4 DNA Ligase+T4 DNA Ligase Buffer+Type IIS Restriction Endonuclease+Backbone Plasmid+ddH2O

    1. Label the 1.5 ml eppendorf tubes and the 0.2 ml eppendorf tubes.

    2. First configure enough MIX according to the amount of experiment requirement.

    3. Add all the reagents to a 0.2 ml eppendorf tube in ice bath.

    4. Blend the reagents in the 0.2 ml eppendorf tube by short centrifugation.

    5. Place the 0.2 ml eppendorf tubes in PCR instrument and run the Golden Gte program.

    Instrument Flow

    Step1: 37℃——5min

    Step2: 16℃——5min

    Step3: 20 times to step1

    Step4: 50℃——5min

    Step5: 80℃——5min

    Step6: 4℃——∞

    Step7: END

    4. Preparetion of Competent Cell

    1. Prepare the sterile Transformation & Storage Solution(TSS)

    Filter the 2*TSS with a 0.22μm filter

    2. Making Competent cell

    Apparatus: test tubes, 50ml eppendorf tubes, 0.5ml eppendorf tubes, 150ml conical flasks, ice box.

    Day 1: Take a DH5α streak on antibiotic-free plates and overnight culture (12-18h).

    Day 2: Pick a colony and inoculate in 5ml LB. Grow the cells overnight (12-16h) at 37℃ and 220 rpm.

    Day 3: Make a 1:100 dilution of overnight culture in LB. Grow the cells at 37 ° C and 225 rpm until the OD600 is about 0.3~0.4.

    Transfer the culture to a clean 50 ml eppendorf tube. Put the tube in ice bath for 30 minutes to cool the culture to 0℃. Centrifugation in 4℃, 1000g for 10 minutes, then discard the supernatant and gather the bacterium.

    Add 2.5 ml pre-cooled LB. Then add 2.5 ml pre-cooled 2*TSS and blend them gently. Put the tube in ice bath and get competent cells 10 minutes later.

    5. Transformation

    1. Label the 1.5 ml eppendorf tubes.

    2. Let competent cells melt naturally in ice bath, and pre-cool the eppendorf tubes.

    3. Blend 50μL competent cells into an eppendorf tube with 5μL Golden Gate products gentlely.

    4. Place the mixed plasmid and the competent cells in ice bath for 30 minutes.

    5. Heat shock 90s in a 42℃ water bath

    5. Remove the water bath, adjust the temperature to 42℃, heat shock 90s, pay attention to timing.

    6. Transfer the tubes quickly back to ice for 10 minutes.

    7. Incubate the cells in 450μL LB medium + Chloramphenical.

    8. Grow the cells at 37 °C and 220rpm for 1 hour

    9. Aseptically spread plate on LB(with Chloramphenical) + Cam plates.

    10. Incubate at 37°C overnight.

    6. Pick a Monoclonal Colony:

    Pick a colony from each of the transformation plates and inoculate in 5mL LB medium + Chloramphenicol. Let the cells grow overnight at 37°C and 220 rpm.

    7. Culture Preservation

    1. Make a 1:1 dilution of each overnight culture in glycerinum (500μL of culture into 500μL of glycerinum).

    2. Store the bacterium in -20℃.

    8. Kit extraction plasmid

    Strictly refer to the instruction manual of the kit and follow the procedure.

    1. Take 1-4 ml of the overnight culture solution, add to the 1.5ml eppendorf tube, and centrifuge for 1 minute at 12,000 rpm (~13,400×g) using a conventional benchtop centrifuge. Try to remove the supernatant as much as possible. (Centrifuge the bacterial pellet into a centrifuge tube and collect the bacterial pellet into a centrifuge tube).

    2. Add 150μL of buffer P1 tao the 1.5ml eppendorf tube containing the bacterial pellet, and completely suspend the bacterial pellet using a pipette or vortex shaker.

    3. Add 150μL of buffer P2 to the 1.5ml eppendorf tube and gently invert it 6-8 times to fully lyse the cells.

    4. Add 350μL of buffer P5 to the 1.5ml eppendorf tube and quickly flip it up and down 12–20 times, mix thoroughly, and a white flocculent precipitate will appear. Centrifuge at 12,000 rpm (~13,400 xg) for 2 minutes at which time a precipitate formed at the bottom of the 1.5ml eppendorf tube.

    5. Transfer the supernatant collected in the previous step to the adsorption column CP3 with a pipette (the adsorption column is placed in the collection tube), taking care not to aspirate the precipitate as much as possible. Centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, drain the liquid from the collection tube, and place the adsorption column CP3 into the collection tube.

    6. Add 300 μL of buffer PWT to the adsorption column CP3 (please check if anhydrous ethanol has been added), centrifuge at 12,000 rpm (~13,400×g) for 30 sec, pour off the waste liquid in the collection tube, and place the adsorption column CP3. Into the collection tube.

    7. Place the adsorption column CP3 in a collection tube and centrifuge at 12,000 rpm (~13,400×g) for 1 minute to remove the residual buffer from the adsorption column.

    8. Place the adsorption column CP3 in a clean centrifuge tube, add 50μL ddH2O to the middle of the adsorption membrane, leave it at room temperature for 3 minutes, centrifuge at 12,000 rpm (~13,400×g) for 30 sec, and collect the plasmid solution into the centrifuge tube.

    9. DNA concentration measurement

    Use NanoDrop2000 measure the plasmid concentration.

    10. Digestion

    Single digestion: 6μL plasmid+0μL EcoRI+0.2μL PstI+1μL Cutsmart+2.8μLddH2O

    Double digestion: 6μL plasmid+0.2μL EcoRI+0.2μL PstI+1μL Cutsmart+2.6μLddH2O

    Digest the plasmid at 37℃ for an hour.

    11. Agarose gel electrophoresis

    1. Add 0.7% agarose to 35ml TAE and dissolve them by microwave.

    2. After the solution cooled, add 7μLEB to 35mlTAE. Blend and pour it into a board. Then insert a sample comb and wait until it form a gel.

    3. Add sample: 3μL marker, 6μL sample. Run at 120V for about 25 minutes.

    12. Fluorescence measurement

    1. Take 100μL glycerol bacteria and inoculate in 5ml LB medium + chloramphenicol in test tube.

    2. Grow the bacteria at 37 °C and 220 rpm until the Abs600 is about 0.1.

    3. Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 8 ml LB medium + Chloramphenicol in test tube.

    4. Add 100μL of each sample to a black 96-well plate, prior to incubation.

    5. Incubate the remainder of the cultures at the target temperature and 220 rpm.

    6. Measure the fluorescence and Abs600. The fluorescence is measured in sensitivity 75. The measurement is protected from light.

    7. At each time point 0 hours, 4 hours, 6 hours, 8 hours and 10 hours, repeat Step 4-6.