Team:NWPU/Protocol

Protocol

1. PCR:

    PCR system: 20μl

Component
Concentration
sterile water 12.7μl
5×Fast pfu Buffer 4μl
dNTP 1.6μl
Forward primer 0.5μl
Reversed primer 0.5μl
TemplateDNA 0.5μl
Fastpfu 0.2μl

    Establish appropriate response procedures on PCR:

    For 4, 5, 6 part:

STEP
TEM
TIME
CYCLES
1 initial denaturation 95°C 10min 1
2 denaturation 95°C 20s 30
3 annealing 58°C 20s
4 extension 72°C 20s 1
5 circulation Repeat the process 2-4 30-35 times
6 extension 74°C 10-15min
7 hold 4°C Hold

    For 1, 2, 3 part:

STEP
TEM
TIME
CYCLES
1 initial denaturation 95°C 10min 1
2 denaturation 95°C 20s 30
3 annealing 58°C 20s
4 extension 72°C 2min 40s 1
5 circulation Repeat the process 2-4 30-35 times
6 extension 74°C 10-15min
7 hold 4°C Pause

2. Agarose gel electrophoresis

    Pouring a Standard 1% Agarose Gel:

  1. Measure out 0.25g of agarose.
  2. Pour agarose powder into microwavable flask along with 25mL of 1xTAE.
  3. Microwave for 1-3min (until the agarose is completely dissolved and there is a nice rolling boil).
  4. Let agarose solution cool down for 5min.
  5. Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5μg/mL (usually about 2-3μl of lab stock solution per 100mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.
  6. Pour the agarose into a gel tray with the well comb in place.
  7. Place newly poured gel at 4°C for 10-15 minutes OR let sit at room temperature for 20-30 minutes, until it has completely solidified.

    Loading Samples and Running an Agarose Gel:

  1. Add loading buffer to each of your digest samples.
  2. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
  3. Fill gel box with 1xTAE (or TBE) until the gel is covered.
  4. Carefully load a molecular weight ladder into the first lane of the gel.
  5. Carefully load your samples into the additional wells of the gel.
  6. Run the gel at 120V until the dye line is approximately 75-80% of the way down the gel.
  7. Turn OFF power, disconnect the electrodes from the power source, and then carefully
  8. Remove the gel from the gel box.
  9. Using any device that has UV light, visualize your DNA fragments.

3. Gel extraction

  1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
  2. Weigh the gel slice in a colorless tube. Add 1 volumes of Buffer QG to 1 volume of gel.
  3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.

4. One step connection and Gibson connection

    With the Gibson assembly method fast assembly of multiple DNA fragments is possible under isothermal conditions, regardless of fragment length.Therefore fragments with overlapping ends of 15-30 bp are needed and can be created via PCR. The Gibson Assembly was performed using the Gibson Assembly® Master Mix according to the Protocol by New England Biolabs [4], unless stated otherwise. To design the primers for the respective PCRs the Software Geneious[5] was used.

  • Set up the reaction according to the table below on ice (2-3 fragment assembly).
  • Incubate samples in a thermocycler at 50 °C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20 °C for subsequent transformation.
  • Transform NEB 5-alpha Competent E.coli cells with 2 μl of the assembly reaction, following the transformation protocol.
Total Amount of Fragments 0.02-0.5 pmols
Gibson Assembly Master Mix (2X) 10 μl
Deionized H2O 10-X μl
Total Volume 20 μl

5. Transformation

  1. Take competent cells out of -80°C and thaw on ice (approximately 20-30min).
  2. Take agar plates (containing the appropriate antibiotic ) out of 4°C to warm up to room temperature or place in 37°C incubator.
  3. Mix 1 to 5μl of DNA (usually 10pg to 100ng) into 20-50μL of competent cells in a microcentrifuge or falcon tube. Gently mix by flicking the bottom of the tube with your finger a few times.
  4. Place the competent cell/DNA mixture on ice for 20-30min.
  5. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 seconds (45sec is usually ideal, but this varies depending on the competent cells you are using).
  6. Put the tubes back on ice for 2 min.
  7. Add 250-500μl LB or SOC media (without antibiotic) and grow in 37°C shaking incubator for 45min.
  8. Plate some or all of the transformation onto a 10cm LB agar plate containing the appropriate antibiotic.
  9. Incubate plates at 37°C overnight.

6. Plasmidextraction

  1. Grow 2mL overnight cultures from single colonies of bacteria containing your plasmid of interest.
  2. Add 1.5mL of the stock culture to a 1.75mL microfuge tube.
  3. Centrifuge in microfuge tube at 10,000g for 30sec.
  4. Pour off the supernatant, being careful not to disturb the bacterial pellet.
  5. Resuspend the pellet in 100μl of cold Solution I.
  6. Vortex the solution for 2 minutes or until all bacteria are fully resuspended.
  7. Add 200μl of Solution II and invert the tube carefully 5 times to mix the contents. The contents will become clear and thicker as the proteins and DNA are denatured.
  8. Incubate solution on ice for 5 minutes.
  9. Add 150μl of cold Solution III to each tube.
  10. Mix by inverting several times. A white precipitate will be formed which contains the bacterial proteins and genomic DNA.
  11. Incubate tube on ice for 5 minutes.
  12. Centrifuge the tube for 5 minutes at 12,000g.
  13. Collect the supernatant into a new tube by pipetting or carefully pouring.
  14. Add either 700μL of cold 100% ethanol or 350μL room temperature isopropanol to the solution to precipitate the plasmid DNA; see detailed protocol below.
  15. Pour out the supernatant.
  16. Air dry the pellet (can be done by inverting the tube at an angle over kimwipe) for 20-30 minutes.
  17. Resuspend pellet with 25-50μl of TE.

7. Induction culture of protein

  1. add bacteria solution in tube to 800 mL 2YT fluid nutrient medium(Kan+,100 μg/mL) (Amp,100μg/mL) 37 °C、220 r/min cultivate intable concentrator until the OD600 is 0.6;
  2. lower the temperature intable concentrator to 16 °C. When the temperature of the bacteria solution decreases, add IPTG to the final concentration 1mM(the best condition when doing the experiment),induction expression16 h;
  3. when the expression is finished,remove the bacteria solution to fungus bottle,5500 r/mincentrifuge15 min;
  4. throw away the supernate,using 25 mL proteinbuffer solution(according to the different properties of different proteins)mix the bacteria sediment,pour into 50 mL centrifuge tube, preserved in the -20°C refrigerator。

8. The experiment of purifying the protein

  1. collect the thallus cell mentioned above, using the high pressure and cryogenic cell cracker,under the pressure of 1200 bar, break the bacteria 2~3 times at 4 °C, in order to make interest protein release from the cell and dissolve into the protein buffer solution. B: use high speed centrifuge(Thermo), at the condition of 4°C、8000 r/min、50min. collect the supernate and the sediments.
  2. suction filtrate the supernate from last step using millipore filterthrough 0.45 μmmicropore. Because the interest protein is on the pET-28a(+)carrier. The N side has a histidine-tag, which allows it to combine with Niaffinity column. Therefore,fusion protein is able to attach to the affinity column. Meanwhile, some mixed protein can stay at the Niaffinity column through non-specific combination, however, the combination is weak. So, by Imidazole gradient elution can we eliminate some mixed protein, reaching the aim of purify the protein. Normally,the process of purifying the protein should be determined after finishing the experiment of testing the conditiong of Imidazole gradient elution. As the following:
    • Column regeneration
    • Use the sample
    • Protein elution

9. SDS-PAGE

    Make the separating gel:

Component\Gel density
8%
10%
12%
15%
ddH2O 2.3mL 1.9mL 1.6mL 1.1mL
30% Acrylamide 1.3mL 1.7mL 2.0mL 2.5mL
1.5M Tris-HCl(pH8.8) 1.3mL 1.3mL 1.3mL 1.3mL
10% SDS 0.05mL 0.05mL 0.05mL 0.05mL
10% Ammonium sulfate 0.05mL 0.05mL 0.05mL 0.05mL
TEMED 0.003mL 0.002mL 0.002mL 0.002mL

    Make the stacking gel:

SDS-PAGE stacking gel(2mL)
component
volume(mL)
ddH2O 1.4
30% Acrylamide 0.33
1.0M Tris-HCl(pH6.8) 0.25
10% SDS 0.02
10% Ammonium sulfate 0.02
TEMED 0.002
  1. Make sure a complete gelation of the stacking gel and take out the comb. Take the glass plates out of the casting frame and set them in the cell buffer dam. Pour the running buffer (electrophoresis buffer) into the inner chamber and keep pouring after overflow until the buffer surface reaches the required level in the outer chamber.
  2. Mix your samples with sample buffer (loading buffer). Heat them in boiling water for 5-10 min.
  3. Load prepared samples into wells and make sure not to overflow. Don't forget loading protein marker into the first lane. Then cover the top and connect the anodes.
  4. Set an appropriate volt and run the electrophoresis when everything's done.
  5. As for the total running time, stop SDS-PAGE running when the downmost sign of the protein marker (if no visible sign, inquire the manufacturer) almost reaches the foot line of the glass plate. Generally, about 1 hour for a 120V voltage and a 12% separating gel. For a separating gel posessing higher percentage of acylamide, the time will be longer.

    Staining & Destaining

  • stain with Coomassie Blue staining solution for half an hour.
  • destain first with destaining solution, then proceed destaining with destilled water.

10. Protein concentration monitoring

    Use ELISA kit to detect the concentration of the concentrated protein.

11. Various ration formula of buffer

     BFD:With all the ultra pure water solution, filtering

IL Buffer A formula
1L Buffer B formula(regulate PH to 7.4)
0.1mol/L K2HPO4 401ml 0.1mol/L K2HPO4 401ml
0.1mol/L K2HPO4 99ml 0.1mol/L K2HPO4 99ml
MgSO4 5mmol MgSO4 5mmol
H2O 500ml H2O Supply to 1L
imidazole 1mol

    Tris buffer With all the ultra pure water solution, filtering

IL Buffer A formula
1L Buffer B formula(regulate PH to 7.4)
Tris 50mmol Tris 50mmol
H2O 1L H2O Supply to 1L
imidazole 1mol

12. Experimental procedure to make reaction

  • Use buffer A with tpp to configurate50×Formaldehyde Solution and dilute it to a certain multiple according to the experimental requirements.
  • Add the pure enzyme or bacteria into it and react in 37°C,whose reaction volume is 200μl.
  • Add BFD enzyme or bacteria ,letting them react for 2h and then add the new enzyme to the bacteria(10OD).
  • After 2 times of such reaction, addtalb pure enzyme or the bacteria(10OD).
  • React for 24h and then add the new enzyme or bacteria.
  • Centrifugal reaction solution and take liquid phase into liquid vial.

13. HPLC

    Put the liquid vials and standard sample into HPLC.Analyze the data after HPLC works out. Use 87h for the liquid phase, 5mmol/lsulphuric acid which has gone through the suction filter ultrasonicfor moving phase.

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