Polymerase Chain Reaction
| 20 μl system | |
|---|---|
| 10Xbuffer | 2 μl |
| dNTP | 1.6 μl |
| Primer1 | 0.6 μl |
| Primer2 | 0.6 μl |
| PCR polymerase enzyme | 1 μl |
| ddH2O | 12.2 μl |
| Template DNA | 2 μl |
Team:DUT China/Experiments
Experiments
Gel electrophoresis
We chose 6Xloading buffer, add 1 μl loading buffer to 5 μl sample. The agarose gel system is as follows.
| 40 ml | |
|---|---|
| TAE | 40 ml |
| agarose | 0.4 g |
Heat to dissolve, add a small amount of dye after cooling, cool and solidify.
Seamless cloning
The target DNA fragment and linearized vector were added to the test tube at a molar ratio of 2:1 for recombination reaction.
| buffer-enzyme(mix) | 15 μl |
| Linearization carrier | X μl |
| PCR fragment | Y μl |
| ddH2O | W μl |
| total | 20 μl |
Incubation and transformation.
Preparation of competent state of Escherichia coli
The second generation of Escherichia coli was cultured to logarithmic growth stage, placed on ice for 10 min and centrifuged at 4000 rpm for 10 min.
Discard the supernatant, resuspend it with precooled CaCl2, place it on ice for 30 min and centrifuge for 10 min.
The supernatant was discarded and the SOC was resuspended to make competent cells.
Heat shock conversion
The connecting products were added to the competent cells, ice bath for 30 min, water bath at 42 °C for 90 s, ice bath for 5 min, LB medium was added, vibrated for 1 h, and coated on a solid plate for culture.
Colony PCR
The colonies were cultured in centrifuge tube and PCR system was configured.
| 20 μl system | |
|---|---|
| 10Xbuffer | 2 μl |
| dNTP | 1.6 μl |
| Primer1 | 0.6 μl |
| Primer2 | 0.6 μl |
| Colony solution | 1 μl |
| ddH2O | 12.2 μl |
| Template DNA | 2 μl |
LB-Media
For one litre of media use
| Peptone | 5 g |
| Yeast extract powder | 2.5 g |
| NaCl | 5 g |
Adjust pH to 7.0 and sterilize at high temperature and high pressure.
DNA purification
DiaSpin Kit
Plasmid Extraction
DiaSpin Kit
Gel extraction
DiaSpin Kit
p-nitrophenyl butyrate (pNPB) Assay
Prepare 10mM pNPB in acetonitrile, 1:4:95 ratio of acetonitrile pNPB solution, 100% ethanol and 50mM Tris-HCL (pH 8), Plate reader, 24 well plate, Cell lysate. Set up 24 well plate by adding cell lysate and controls. Add the pNPB buffer immediately before running the plate reader. Run the plate at 37 °C for 10 min inside the plate reader, taking measurements of 405 nm absorbance every 15 seconds. 405 nm is the absorbance of pNPB substrate that has been broken by an esterase enzyme, so a significant read at 405 nm absorbance indicates enzyme is breaking ester bonds in the substrate.
Protein re-naturation
The bacterial culture was centrifuged at 5,000 rpm, 4 °C for 15 min before the supernatant was removed, and then the pellets were resuspended in PBS, and sonicated on ice 10 min at 50% duty cycle. Inclusion bodies and cellular debris were collected by centrifugation at 12,000 rpm for 15 min at 4 °C. Then the pellets were resuspended in 50 mL washing buffer (20 mM Tris-HCl, 0.5 M NaCl, 2 M urea, 2% Triton, pH 8.0) for 30 min on ice. The solution was centrifuged at 12,000 rpm for 15 min at 4 °C and then the pellet was resuspended in 50 mL denaturation lysis buffer (20 mM Tris-HCl, 0.5 M NaCl, 8 M urea, 0.2 mM DTT, pH 8.0) and incubated in ice bath for 5 h to completely dissolve the proteins. The insoluble cellular components were removed after centrifugation at 12,000 rpm at 4 °C for 20 min, and the supernatant was filtered using 0.45 μm membrane filters to obtain the protein. Then, the supernantant containing the target protein solution was successively placed into beakers either containing 500 mL dialysis buffer I (20 mM Tris-HCl, 0.5 M NaCl, 8 M urea, pH 8.0), dialysis buffer II (20 mM Tris-HCl, 0.5 M NaCl, 6 M urea, pH 8.0), dialysis buffer III (20 mM Tris-HCl, 0.5 M NaCl, 4 M urea, 0.1 mM Oxidized Glutathione[GSSG], 0.9 mM glutathione[GSH], pH 8.0), dialysis buffer IV (20 mM Tris-HCl, 0.5 M NaCl, 3 M urea, 0.1 mM GSSG, 0.9 mM GSH, pH 8.0), dialysis buffer V (20 mM Tris-HCl, 0.2 M NaCl, 2 M urea, 0.1 mM GSSG, 0.9 mM GSH, pH 8.0), dialysis buffer VI (20 mM Tris-HCl, 0.1 M NaCl, 0 M urea, 0.1 mM GSSG, 0.9 mM GSH, pH 8.0), dialysis buffer VII (20 mM Tris-HCl, 0.1 M NaCl, pH 8.0), dialysis bufferVII(20 mM Tris-HCl, 0.9% NaCl, pH 8.0), and stirred at 4 °C for 4 h.
Western blot
Take the supernatant for SDS-PAGE electrophoresis (70 V 30 min, 110 V 2 h);
After the electrophoresis, cut off the protein gel block, place it in a protein semi-dry transfer instrument, and transfer the target protein to the PVDF membrane under the condition of 15 V 13 min; Put the PVDF membrane into 50 g/L skimmed milk powder and leave it at room temperature for 2 hours (slowly shake); Use 20 g/L skimmed milk powder to dilute the anti-His mouse antibody at a ratio of 1:2000, and add an appropriate amount of the diluted antibody. Submerge the PVDF membrane in a self-made closed bag containing PVDF membrane, and incubate overnight at 4 °C (slowly shaking); Take out the PVDF membrane from the primary antibody solution and place it in a petri dish, wash with TBST 5 times, each time 15 min; Use 20 g/L skimmed milk powder to dilute goat anti-mouse IgG (called secondary antibody) at a ratio of 1:3000, and add an appropriate amount of diluted secondary antibody to a self-made closed bag with PVDF membrane to submerge the PVDF membrane and incubated at room temperature for 2 hours (slowly shake); Then, take out the PVDF membrane from the secondary antibody solution and place it in a plate, wash five times with TBST, 15 minutes each time; After washing, dry the PVDF membrane and drop it on the front. Add the prepared color developer until it is completely covered, place it at room temperature for 2 minutes, place it in an ECL luminescence imager for exposure and photographing, and record the result.