Team:BUCT-China/Experiments
Experiments
Experiments
Reagent
Step
Take 25 sterile test tubes, mark them from 0 to 24.
Incubate the seed solution for 12 h, transfer 5 ml of seed solution to 100 ml of the seed culture medium in a 5% inoculum, and shake the bacteria. The solution was evenly mixed, and 5 ml of the bacterial solution was transferred to 25 tubes.
25 tubes were inoculated at 37°C, l80 rpm in a constant temperature shaker, and 1h as a cycle.The incubation time is set to 0, 1, 2, 3, 4, 5, 6. 7, 8-24h, the sample taken was stored at 4°C, and all samples were taken to determine the absorbance.
Blank seed culture was measured as absorbance at 600 nm wavelength as an experimental control. value. Determine the measurement from the earliest taken out of the test tube, dilute the test tube with a large concentration of the bacterial solution in the medium, and record the dilution factor to ensure that the absorbance is between 0.1 and 0.65.
Reagent
Gly-HCl buffer: Prepare a Gly solution with a final concentration of 20 mM, and adjust the pH value with HCl to obtain a buffer of pH 1.0, 1.5, 2.0, 2.5, 3.0.
NaAc-HAc buffer : A final concentration of 20 mM NaAc solution was prepared, and the pH was adjusted with glacial acetic acid to obtain a buffer having a pH value of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5.
Step
Oxidase was separately added to a buffer solution, and the optimum reaction pH of oxidase was measured at 50℃.
Reagent
50mMl glucose / 10mMl EDTA / 25mMl Tris-HCl, pH = 8.0
0.2Ml NaOH / 1% SDS
3Ml potassium acetate / 2Ml acetic acid
Isopropyl alcohol, Anhydrous ethanol, 75% ethanol should be pre-cooled in -20°C refrigerator; phenol chloroform pre-stored in 4°Crefrigerator
Shake bacteria, 2-3 ml corresponding resistant LB medium, cultured at 37°Covernight
Step
Centrifuge the solution at 12000 rcf for 5 min, discard the supernatant, add 0.2 ml of solution I (already added RNase), mix well, add 0.25 ml of solution II, mix by inversion, place at room temperature for 5 min, add 0.4 ml of solution III After mixing, invert and centrifuge at 13000 rcf for 30 min
Transfer the supernatant to a new 1.5 ml EP tube, add an equal volume of phenol chloroform mixture, and mix well. Centrifuge at 10000rcf for 10 min.
Take the supernatant, transfer to a new EP tube, add an equal volume of pre-chilled isopropanol, mix well, and centrifuge at 13000 rcf for 6 min.
Pour off the supernatant, use 1 ml of pre-cooled 75% ethanol, invert, and centrifuge at 13,000 rcf for 1 min.
Pour off the supernatant and add 1 ml of pre-cooled 75% ethanol along the wall and pour directly.
Add 1 ml of pre-cooled ethanol along the wall and pour directly. After depressing for 5 min, it was left at room temperature for 10 min. After the liquid in the tube is cleaned, 100 ul of water is added to the tube and incubated at 55°C for 5 min.
The resulting plasmid solution can be stored at -20°C for long-term storage.
Reagents and instruments
Acetate-Sodium acetate Buffer 960l (20mM, pH4.5)
ABTS9 (0.5M) 20ml
Crude enzyme solution 20ml
UV-2700 spectrophotometer
Step
The reaction is carried out at 45°C, zero with buffer, The substrate was separately added to the substrate to self-decompose, and then the enzyme solution was added to measure the change in absorbance in one minute.
The enzyme activity was calculated according to the formula U(/mg) = ΔA×V total×N / (ε×t ×V enzyme×d).
△A: change in absorbance per unit time
V total: total activity of enzyme activity measurement (mL)
N: dilution factor of enzyme solution
ε: molar absorptivity
t: reaction time (min)
V enzyme: amount of enzyme added in the reaction system ( mL)
d: cuvette diameter (cm)
Reagent
LB medium
Selective LB agar culture plate
1‰ of antibiotics
Host bacteria: Competent bacteria DH5αtreated with 100 mmol/L CaCl2
Step
Take out the receptive state of large intestine and put it into the ice box at -80℃.
Plasmid 10 (2) ul was added to the TOP10 (BL21) competent state and ice bath was performed for 30min.
Heat excitation was performed at 42℃ and 90s.
Place in the ice box and let stand for 5 minutes.
Add 500ul LB medium to the centrifuge tube.
Culture in a shaking table with 180rpm at 37℃ for 60min.
Add 1‰ of antibiotics into LB tube and culture with 1% bacterial liquid for 12-14h.
Reagent
Standard protein mixture
30% gel stock solution
Separation gel buffer (1.5 mol/L)
Concentrated gel buffer (0.5 mol/L)
Electrode buffer (pH 8.3)
10% SDS
Mass concentration 10% ammonium persulfate (fresh formulation)
Loading buffer
0.25% Coomassie Brilliant Blue R-250 staining solution
Decolorizing solution
Protein sample of unknown molecular weight (1 mg/mL)
Step
Loading plate
Gel polymerization
Configure 10% separation gel and 4.8% concentrated gel.
Configure 10% separation gel and 4.8% concentrated gel.
The sample used in this experiment is a 15 to 20 g standard protein sample solution, placed in a 0.5 mL centrifuge tube, and 15-20 l of the sample treatment solution is added. Treated in a 100°C water bath for 2 min, cooled to room temperature and set aside. A sample of 201 of a protein of unknown molecular weight was aspirated and treated according to the standard protein treatment method.
Loading
Add a sample to each sample tank in a micro-syringe, add 10 to 15 l each (containing 10 to 15 g of protein), and add 20 to 30 l to a dilute solution.
Electrophoresis
The current before the sample is injected is controlled at 15-20 mA, about 15-20 min; after the bromophenol blue indicator in the sample reaches the separation gel, the current rises to 30-45 mA, and the electrophoresis process keeps the current stable. When the bromophenol blue indicator migrates to a distance of 1 to 2 cm from the leading edge, electrophoresis is stopped for about 1-2 hours.
Dyeing, bleaching
The center of the indicator zone was inserted with copper wire as a marker, and stained in a large petri dish, stained with 0.25% Coomassie blue dye solution for 2 to 4 hours. Discard the staining solution, rinse the surface with distilled water several times, then add the decolorizing solution to diffuse and decolorize, often changing the decolorizing solution until the protein band is clear.