Team:CIEI-BJ/Protocol
1. Mix 0.25 gram of agarose with 25 grams of TAE solution, and the heat the mixture until the agarose fully dissolves.
2. Pour the mixture into a square mould. Plug in the comb, and wait until the mixture solidifies.
3. Put the gel into the electrophoresis apparatus, and the fill it with TAE solution.
4. Add 1 μL of loading buffer and 1 μL of SYBR Gold into 5 μL of sample.
5. Add the sample and marker into the gel. Connect two sides with the electric source. Wait for 30-40 minutes.
6. Take out the gel, and observe the result under UV transillumination.
1.Prepare the cells
(1)Prepare 5mL Pichia pastoris in a conical and oscillate it on the oscillating table for several hours.
(2)Put 0.5mL of the Pichia pastoris in the conical. Let it grow to an OD600=1.3-1.5.
(3)Centrifuge the Pichia pastoris at 1500 ×g for 5 minutes in a 4℃ condition. Add 500 mL sterile water (which is previously stored in the ice).
(4)Centrifuge the Pichia pastoris then add 250 mL ice-cold water.
(5)Centrifuge the cells then add 50 mL 1M ice-cold sorbitol.
(6)Centrifuge the cells then add 1.5 mL 1M ice-cold sorbitol.
2.Transformation
(1)Put 80 L Pichia pastoris cells we prepared in previous process in a centrifugal tube and add 5-20 μg of linearized DNA (which we got from the previous experiment)
(2)Transfer the mixture in a particular tube prepared for the eletroporator. Use the electroporator to transform the DNA in the Pichia pastoris.
| 10×Loading Buffer | 2.5μL |
| Ligase | 2.5μL |
| AD plasmid fragment | 10μL |
| BD SOS fragment | 10μL |
| Total | 25μL |
1.Prepare the mixture of the materials above in a 0.2 mL microfuge tube.
2.Incubate the solution at 4 ℃ for overnight or at 16 ℃ for 2-3 hrs.
| BL Buffer | 500μL |
| PB Buffer | 250μL |
| PW Buffer (with 100% ethyl alcohol) | 600μL |
| EB Buffer | 40μL |
| PCR product system | 50μL |
1. Add BL buffer into spin column CB2 (with collection tube 2mL) and centrifuge for 1 min at 12000 rpm. Remove the discard in the collection tube.
2. Add the PCR product and the PB buffer in the CB2 and mix them evenly.
(notice: If the PCR product system is , add 5 PB buffer)
3. Leave the solution for 2 minutes and centrifuge for 1 minute at 12000 rpm. Remove the discard in the collection tube.
(notice: The max volume of column is 800μL, if sample volume is more than 800μL, you can repeat step 3)
4. Add 600μl of Buffer PW to columns CB2. Centrifuge at 12,000rpm for 30-60 sec in a microfuge. Pour the discard solution in tubes; put columns CB2 back to tube. Store the tube at room temperature for 2-5min.
5. Repeat step 4.
6. Centrifuge at 12,000rpm for 2min, pour the discard solution. Store the tube at room temperature for 5min, leaving it as dry as possible.
7. Centrifuge at 12,000rpm for 2min, pour the discard solution. Store the tube at room temperature for 5min, leaving it as dry as possible. Take the DNA solution in the collection tube to carry out gel electrophoresis.
Prepare the reaction mix in PCR tube:
| Template DNA | 1 μL |
| Enzyme(prime STAR) | 1 μL |
| Forward Primer | 2 μL |
| Reverse Primer | 2 μL |
| Easy Jag Mix | 25 μL |
| ddH2O | 19 μL |
Note: We use two types of template DNA 1.5 μL respectively and add ddH2O up to 50 μL.
1.Set the program of thermal cycle:
| 1x | 95 | 5 min |
| 30x | 95 | 30 s |
| 30x | 55 | 30 s |
| 30x | 72 | 1.5 min |
| 1x | 72 | 10 min |
| 1x | 16 | 15 min |
| Template | 1400 μl*2 |
| P1 | 1 μL |
| P2 | 2 μL |
| P3 | 2 μL |
| Washing Buffer | 19 μL |
| Elution Buffer | 1 μL |
Note: Templates include carrier with goal gene (gltB, ScTPS1 and SpTPS1)
1.Pour 1400 μL of the culture into a microfuge tube. Centrifuge at 12,000 rpm for 1 min in a microfuge. Pour the discard solution, leaving it as dry as possible.
2.Repeat step 1
3.Add 400 μL solution P1, wait until all deposition dissolves.
4.Add 400 μL solution P2; mix the content by inverting the tube rapidly five times.
5.Add 560 μL solution P3, wait until all deposition dissolves.
6.Centrifuge the microfuge at 12,000 rpm for 10 min. Transfer the supernatant into a fresh column that in a tube.
7.Centrifuge the tube at 12,000 rpm for 1 min in a microfuge.
8.Add 700 μL Washing buffer (PB), then centrifuge at 12,000rpm for 1 min. Pour the buffer.
9.Add 500 μL Washing buffer (PB), then centrifuge at 12,000rpm for 1 min. Pour the buffer.
10.Centrifuge the tube at 12,000 rpm for 2 min, then open and dry it.
11.Transfer the column to a fresh microfuge tube, then add 80μL Elution buffer (EB), store it at room temperature for 5 min.
12.Centrifuge at 12,000rpm for 1 min. Discard the column.
1.LB:
| Materials | Amount |
| Pichia pastoris Extract | 5 g |
| Tryptone | 10 g |
| NaCl | 10 g |
| ddH2O | To 1 L |
| Ampicillin (20 μg/mL in H2O) |
(1) Add the materials above in a conical flask.
(2) Shake the conical flask till dissolved.
(3) Autoclave the conical flask for 15 min at 15 psi to sterilize on liquid cycle.
2 LB agar
| Materials | Amount |
| Pichia pastoris Extract | 5g |
| Tryptone | 10g |
| NaCl | 10g |
| Agar | 15g |
| ddH2O | To 1L |
(1)Add the materials above in a conical flask.
(2)Shake the conical flask till dissolved.
(3)Autoclave the conical flask for 15 min at 15 psi to sterilize on a liquid cycle.
(4)Then swirl the conical flask gently. After it cool down to 50~60°C, add antibiotics, Ampicillin. Swirl the flask to mix them up.
(5)Pour the LB agar in the conical flask on plates before it solidifies
1.Mix the following substances in a microfuge tube
| SCTPS | SPTPS | GLTB tube | |
| Target Plasmid/μL | 25 | 25 | 25 |
| Xba/μL | 2 | 2 | 2 |
| Spe/μL | 2 | 2 | 2 |
| Buffer/μL | 5 | 5 | 5 |
| ddH2O/μL | 16 | 16 | 16 |
| Total/μL | 50 | 50 | 50 |
2.Set all tubes in the incubator for 2 hours at 37
Materials:
| E. coli-contain solution | 30 μL |
| Test gene | 1μL |
| LB liquid | 500 |
1.Prepare a centrifuge tube with 30μL E. coli-contain solution each.
2.Put under-tested genes in the centrifuge tube with the E. coli-contain solution and mark the tube clearly.
3.Put the tube on the ice for 30 min.
4.Put the centrifuge tube in the 42°C water-bath.
5.Put the tube back to ice for 2 min.
6.Put 500 μL LB culture-medium into tube and put them into the shaker for an hour at 200rpm, 37°C.
7.Take 500 μL solution from the tube and spread it on ampicillin-contain culture dishes.
8.Put the dish into the incubator at 37°C.
1.Place cell culture plate on ice. Wash the cells with ice-cold PBS
2.Discard the PBS solution, then add ice-cold lysis buffer
3.Scrape the cells off the plate with a cold cell scraper. Transfer the cell suspensions to a cool micro centrifuge tube.
4.Keep the micro centrifuge tube with cell suspension at 4℃ for 30 minutes
5.Centrifuge at 4℃ for 12,000 rpm for 20 minutes
6.Remove the tubes from and place them on ice. Transfer the supernatant in a fresh tube on ice. Discard the pellet.
7.Take a small volume of lysate to determine the quantity of protein. Determine the concentration of each sample of lysate.
8.Take a predetermined quantity of protein, and add equal volume of loading buffer.
9.Boil each sample with loading buffer at 100℃ for 5 minutes to denature the protein.
10.Load equal amount of protein and molecular weight marker into wells of SDS-PAGE gel.
11.Run the gel at 100v until the proteins are separated completely.
12.Activate the PVDF membrane with methanol for 1 minute and rinse with transfer buffer.
13.Prepare a stack as follows:
Positive side of apparatus; Sponge; Filter paper; Membrane; Gel; Filter paper; Sponge; Negative side of apparatus
14.Block the membrane for 1 hour at room temperature with blocking buffer
15.Incubate the membrane with primary antibody in blocking buffer at 4 ℃ overnight.
16.Wash the membrane using TBST for 3 times. Wash for 5 minutes each time.
17.Incubate the membrane with secondary antibody in blocking buffer at room temperature for 1 hour
18.Repeat procedure 16.
19.Remove excessive reagent. Put the membrane in transparent plastic sheets
20.Acquire image with apparatus.
香茅醇提取和检测方法
