Team:GDSYZX/protocal
PROTOCAL
1 Synthesis of HQT (1320bp)
HQT was synthesized in Guangzhou TIANYI HUIYUAN Technology Co. Ltd. Before synthesis, we performed codon optimization according to the codon preference of Oryza sativa L. and removed the illegal restriction sites.
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2 Vector Construction
2.1 Enzyme Digestion of PUC19-HA
| Reagent | Volume |
|---|---|
| 10 x Cutsmart buffer | 5 μL |
| BamHI | 0.5 μL |
| StuI | 0.5 μL |
| DNA | 1 μL |
| ddH2O | 43 μL |
P.S. Plasmid template : about 800 ng (1 μL). React condition: 37°C，8-10 h.
2.2 Clone Target Segments of HQT and GluD-1
Use PCR to clone the target segment of gene HQT and GluD-1 promoter. Here ‘s the react condition:
| Reagent | Volume |
|---|---|
| Q5 DNA polymerase | 0.5 μL |
| 5 × Q5 buffer | 10 μL |
| 10 mM dNTPs | 1 μL |
| Upstream primer | 2.5 μL |
| Downstream primer | 2.5 μL |
| template | 1 μL |
| ddH2O | 32.5 μL |
P.S. the concentration of template is 1 ng/ΜL.
Reaction program:
| Temperature (°C) | Lasting Time | |
|---|---|---|
| Initiation | 98 °C | 30sec |
| Cycles 35X | 98 °C 55 °C 72 °C | 10sec 30sec 20-30sec/kb |
| Final Extension | 72 °C | 2 min |
P.S. annealing temperature and extending time depends on the Tm value of primers and the size of target segment.
2.3 DNA Gel Recycling
(1)Respectively prepare 6 M NaI and DNA washing solution(see appendix for formulation)；
(2) Add 300μL 6M NaI to the gel containing the target DNA band ( The metal bath is heated at 70°C until the gel is completely melted (about 5-10min).
(3) Add 10μL SiO2 (5 g/30 mL) to the melted mixture. Use the instrument of vortex to mix the solution, then kept them at room temperature for 2min.
(4)Centrifuge the solution for 20 s with the speed of 14000 rpm at room temperature, then use the vacuum pump to remove supernatant.
(5)Add 500 μ LDNA washing buffer solution to the precipitate, then blow and mix the precipitate with a pipettor and centrifuge for 20s with the speed of 14000 rpm at room temperature, then use the vacuum pump to remove the supernatant. This step is repeated twice.
(6)Centrifuge for 1 min with the speed of 14000 rpm at room temperature, then use vacuum pump to suck out the supernatant liquid
(7) Use the metal bath to heat the solution at 70°C for 30 s, add 10 μL / 45 μL of ultra-pure water sterilization to the precipitation, liquid transfer gun blow mixing precipitation.
(8)Use the metal bath to heat the solution at 70°C for 1min. Centrifuge for 2 min with the speed of 14000 rpm at room temperature. transfer supernatant to new centrifuge tube.
2.4 Enzyme Digestion of Target Segments
| Reagent | Volume |
|---|---|
| 10 x Cutsmart Buffer | 5 μL |
| BamHI | 0.5 μL |
| StuI | 0.5 μL |
| DNA | 44 μL |
P.S.: template from PCR product recovery (44μL). Conditions of reaction :37°C,8-10h.
2.5 Link Vectors And Target Segments
| Reagent | Volume |
|---|---|
| recombinase | 1 μL |
| buffer | 2 μL |
| vectors | 2 μL |
| Target gene | 1 μL |
| ddH2O | 4 μL |
P.S.: Reaction conditions :37°C,30min.
2.6 transform recombined vector into E.coli
(1) Add 10 microliters of ligation product to 50 microliters of receptive E-coli, use the pipette to beat upon it well and place it on the ice for 30 min.
(2) Heat the tube in a water bath at 42°C for 30 s and place it on the ice for 3-5 minutes.
(3)Add 500 μL of LB sterilization medium (without antibiotics) to centrifuge tube and mix well, then incubate the bacteria at 37℃ 200rpm for 1 hour.
(4) Centrifuge for 1 min under room temperature (5000rpm), then pour the supernatant away till there are about 100μL fungi left.
(5)Re-suspend the bacteria and coat them in LB solid medium that contains ampicillin for 10-12 h at 37°C.
2.7 Selection of Positive Clones
2.7.1 PCR verification of bacterial solution
Sixteen colonies growing on LB solid medium containing ampicillin were selected and verified by 2x Easy Taq enzyme. System as follows:
| Reagent | Volume |
|---|---|
| 2x Easy Taq Mix | 10 μL |
| bacterial colony | 0 |
| Upstream primer | 1 μL |
| Downstream primer | 1 μL |
| ddH2O | 8 μL |
Reaction condition:
| Temperature (°C) | Lasting Time | |
|---|---|---|
| Initiation | 98 °C | 30sec |
| Cycles 35X | 98 °C 55 °C 72 °C | 10sec 30sec 20-30sec/kb |
| Final Extension | 72 °C | 2 min |
Note: Annealing temperature and elongation time are determined by primer Tm value and target gene length.
2.7.2 sequence verification
The right clone was confirmed by PCR. The LB medium with ampicillin was used to amplify the culture at 37 ° C for 10-12 H. The strain was preserved with 50% glycerol (V50% glycerol / V bacterial solution = 1:1). Then, the plasmids were extracted with the Plasmid Extraction Kit. After measuring the concentration, about 10 μL was taken and sent to the sequencing company for sequencing. The sequenced plasmid was stored at - 20 ° C, and the corresponding bacterial solution was stored at - 80 ° C.
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3 massive plasmid extraction
(1) Cultivating bacteria. Inoculate the bacteria in sterile TB medium with 0.5% volume of glycerol and antibiotics. Cultivate the bacteria at 37°C and 220 rpm. After about 12 hours, the bacteria solution will reach a saturated state. (Bacteria can be frozen at -20°C if not used temporarily).
(2) Collect bacteria. Centrifuge at 10,000 rpm, room temperature for 6 min to collect the bacteria, discard the supernatant, and remove the remaining supernatant with absorbent paper (the bacteria that are not in use can be stored at -20°C).
(3) Resuspend the bacteria. Add 10 mL solution I to resuspend the bacteria, and then add 100 μL RNAase (10 mg/mL) to reduce the RNA content in the plasmid.
(4) Add 10 mL of solution II and gently invert the bacterial solution up and down 8 times. At this time, you can see that the bacterial solution looks like egg white.
(5) Add 10 mL of solution III and slowly invert the bacterial solution 10 times. At this time, a white viscous precipitate can be seen.
(6) Centrifuge for 10 min at 10000 rpm, room temperature. Dispense the supernatant into about 20 1.5 mL centrifuge tubes, centrifuge at 14000 rpm at room temperature for 10 min. Then combine the supernatant into a 50 mL centrifuge tube.
(7) Add 0.6 times the volume of isopropanol to the supernatant, and invert the tube about 10 times until the solution is uniform.
(8) Add the mixed solution to 12 DNA binding columns equilibrated with CBS solution, and centrifuge at 10,000 rpm at room temperature for 15 s. Repeat this step until the solution is all over.
(9) Add 500 μL of W1 solution to each DNA binding column, centrifuge at 10000 rpm, room temperature for 15 s, and discard the column solution.
(10) Add 500 μL Wash solution to each DNA binding column, centrifuge at 10000 rpm, room temperature for 15 s, and discard the column solution.
(11) Repeat step 10.
(12) Centrifuge for 2 min at 12000 rpm, room temperature.
(13) Replace the tube under the DNA binding column with a new 1.5 mL centrifuge tube, and place the opening in a 70°C metal bath to heat for 2 minutes.
(14) Add sterile ultrapure water (approximately 50 μL) to each DNA binding column, heat it in a metal bath at 70°C for 2 minutes, 14000 rpm, room temperature, centrifuge for 3 minutes, and combine the plasmids into one tube. Determine the plasmid concentration, DNA gel electrophoresis to detect RNA content, enzyme digestion or sequencing to identify whether the plasmid is correct.
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4 Preparation of rice protoplasts
(1) The enzymatic hydrolysate was prepared (see appendix table for detailed formula). The impurities were removed from the enzymatic hydrolysate filtered with a 0.22 μm filter and the enzymatic hydrolysate was poured into a 10cm cell culture dish for later use.
(2) Seven-10d rice seedlings with good growth were selected, and the stems and sheath tissues of 40-60 rice seedlings were cut into strips of about 0.5mm, which were quickly placed in the cell culture dish containing the enzymatic hydrolysate of leaves. The strips were dispersed, completely immersed in the enzymatic hydrolysate, and shaken gently in the dark for 3 h.
(3) The cell culture dishes were placed in a horizontal shaker at 60 RPM, shaken at room temperature and away from light for 3 min, to fully release the protoplasts in the leaves.
(4) Equal volume of W5 solution was added to the cell culture dish (see appendix table for detailed formula), and the culture dish was gently shaken to fully release the protoplasts.
(5) 40 m nylon mesh was used to filter the protoplast enzymatic hydrolysate into 30 mL circular bottom centrifuge tube to remove the impurities of the protoplast enzymatic hydrolysate.
(6) The horizontal centrifuge was centrifuged at 240 RPM for 5 min at room temperature, and the supernatant was removed by vacuum pump.
(7) the protoplasts were resuspended by adding W5 solution of the same volume as the enzymatic hydrolysate, and the protoplasts were placed on ice for rest for 30min.
(8) 1 mL 5% serum was added to each well of the 6-well plate, the bottom was pressed, the 6-well plate was gently rotated, so that the serum completely covered the bottom of the 6-well plate, the serum was sucked, and 1 mL protoplast culture medium WI was added to each well (see appendix table for detailed formula).
(9) In step 7, the protoplasts rested on the ice, and were centrifuged in a horizontal centrifuge at 240 RPM for 5 min at room temperature. The supernatant was removed with a vacuum pump, and the protoplasm was resuspended by adding MMg of the same volume (see the appendix table for detailed formula)
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5 Transfection of rice protoplasts
(1) A plasmid with a total volume of 20 μL and a concentration of 2.2 ug/μL was added to 2 mL circular bottom centrifuge tube.
(2) Rice protoplasts prepared from 200 L were added into the above 2 mL centrifuge tube. After that, 220 μL 50% PEG 4000 solution was added immediately. The solution was mixed with finger flicking and transfected in dark at room temperature for 15 min.
(3) 800μL W5 solution was added to each 2 mL centrifuge tube, and the transfection was stopped by gently turning upside down twice.
(4) Horizontal centrifuge, 280 RPM, centrifuge for 5 min at room temperature.Vacuum pumps remove the supernatant until the final volume is about 30 μL.
(5) 100μL W5 was added along the wall of the 2 mL centrifuge tube. The cells were then transferred to a 6-well plate of the refueling medium.
(6) Gently rotate the 6-well plate to evenly distribute the protoplasmic cells in the culture medium.
(7) Incubation in darkness for 6-12 h at room temperature (the specific time is determined according to the experiment).
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6 Western blot
(1) The preparation methods of 10% (15%) polyacrylamide gel, SDS-PAGE buffer and Transfer buffer are shown in the appendix.
(2) The samples were added into the wells of the polyacrylamide gel in a certain order, and then the protein electrophoresis was carried out at a voltage of 100 V for about 2 h.
(3) After the electrophoresis, cut the PVDF membrane (the size depends on needs), immerse the PVDF membrane in methanol for 30 s to activate it, and then wash the membrane with pure water for 3-4 times.
(4) Put them in the sequence of filter paper, gel, PVDF membrane and filter paper. The transfer current was controlled at 350 mA for 2-3 h.
(5) After the membrane transfer, the PVDF membrane was immersed in 5% skim milk powder solution (1 g/20 mLTBST buffer solution) in a horizontal shaper at 60 RPM, room temperature, and sealed for 1 h.
(6) Add 1 L antibody to the milk, continue to incubate for 2-4 h, discard the milk.
(7) Wash the film with 1 X TBST buffer solution for 4 times, each interval of 15 min. Finally, all the solutions are poured out, and the chromogenic solution is added to take pictures under the imager.
(8) For the membranes requiring secondary antibody incubation, 1 X TBST buffer can be poured out after the membrane is washed at step (7), and 5% skim milk solution containing secondary antibody can be added in a horizontal shaper at 60 RPM for further incubation for 2-4 h.
(9) Same as Step 7.
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7 Extractionand Detection of Purpose Products
7.1 Obtained Cells Expressing The Target Gene
Centrifugated (1000 rpm, room temperature) for 2 minutes, kept 30 μL of the supernatant，and removed others.
7.2 Cell Disruption
The cells obtained in the previous step were cleaved by a vortex oscillator, centrifuged for 10 minutes(13,000 rpm, room temperature), and the supernatant was collected into another 2 mL centrifugal tube.
7.3 Detection of Target Product by Liquid Chromatography
Conditions for liquid chromatography:
Mobile Phase A: water
Mobile Phase B: Acetonitrile
The flowrate was 1 mL/min and the elution time was 60 min.
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Appendix
| Reagent | Final Concentration | Dosage |
|---|---|---|
| 0.5 M EDTA | 10 mM | 10 mL |
| 1 M Tris-HCl（pH7.5） | 50 mM | 25 mL |
| RNase A powder | 100 μg/ml | 100 mg |
| dH2O | Add to 500 mL |
| Reagent | Final Concentration | Dosage |
|---|---|---|
| 20% SDS | 1%（w/v） | 50 mL |
| 5 M NaOH | 0.2 M | 40 mL |
| dH2O | Add to 1L |
| Reagent | Dosage |
|---|---|
| CH3COOK | 64.75 g |
| CH3COOH | About 45mL |
| dH2O | Add to 500 mL |
| Reagent | Final Concentration | Dosage |
|---|---|---|
| Macerozym R10 | 0.4%（w/v） | 0.04g |
| Cellulase R10 | 1.5%（w/v） | 0.15g |
| 2 M KCl | 20 mM | 0.1 mL |
| 0.2 M MES | 20 mM | 1 mL |
| 0.8 M mannitol | 0.4 M | 5 mL |
| 1 M CaCl2 | 10 mM | 0.1 mL |
Dissolveina55°Cwaterbath， 10min； aftercoolingontheice, addthefollowing reagentinproportion 55°C
| Reagent | Final Concentration | Dosage |
|---|---|---|
| 10% bovine serum albumin（BSA） | 0.1%（w/v） | 0.1 mL |
| dH2O | 3.7 mL |
| Reagent | Final Concentration | Dosage |
|---|---|---|
| 0.2 M MES | 4 mM | 20 mL |
| 2 M KCl | 20 mM | 10 mL |
| 0.8 M mannitol | 0.5 M | 625 mL |
| dH2O | Add to 500 mL |
| Reagent | Final Concentration | Dosage |
|---|---|---|
| 2 M KCl | 5 mM | 2.5 mL |
| 1 M CaCl2 | 125 mM | 125 mL |
| 5 M NaCl | 154 mM | 30.8 mL |
| 0.2 M MES | 2 mM | 10 mL |
| dH2O | Add to 1 L |
| Reagent | Final Concentration | Dosage |
|---|---|---|
| PEG4000 | 40%（w/v） | 8 g |
| 1 M CaCl2 | 100 mM | 2 mL |
| 0.8 M mannitol | 0.2 M | 5 mL |
| dH2O | Add to 20 mL |
| Reagent | Final Concentration | Dosage |
|---|---|---|
| 0.2 M MES | 4 mM | 10 mL |
| 2 M MgCl2 | 15 mM | 3.75 mL |
| 0.8 M mannitol | 0.4 M | 250 mL |
| dH2O | Add to 500 mL |
| Reagent | Dosage |
|---|---|
| Glycine | 72 g |
| Tris-base | 15.1 g |
| SDS | 5 g |
| dH2O | Add to 500 mL |
| Reagent | Dosage |
|---|---|
| Tris-base | 15.1 g |
| Glycine | 72 g |
| dH2O | Add to 500 mL |
Dilute ten times to use, add 100 ml methanol to every 1 L of 1xTransfer buffer
| Reagent | Final Concentration |
|---|---|
| Tris-base（trihydroxymethyl aminomethane） | 2 M |
| Glacial acetic acid | 1 M |
| EDTA | 50 mM |
pH8.2，purchase from Shanghai Bioscience Technology Co.Ltd
| Reagent | Final Concentration |
|---|---|
| Tween-20 | 0.5% |
| NaCl | 1.5 M |
| Tris-HCl | 250 mM |
pH7.5，purchase from Shanghai Bioscience Technology Co.Ltd