Team:GDSYZX/protocal
PROTOCAL
Experimental note 6.3-6.19
Transcriptome analysis was done.
Experimental note 6.20-7.2
Pset152 and promoter recombination.
Experimental note 6.27-7.3
The promoter was recombined with eGFP.
Experimental note 7.4-7.10
Binder identification.
Experimental note 7.11-7.18
The binder was inoculated into LB, and the fluorescence intensity is measured. P24880 with a fluorescence intensity of 868 has the highest fluorescence intensity.
Experimental note 7.20.2021
PCR operation Pro24880 (the strongest promoter that has been screened out).
| Total | 50 μl |
|---|---|
| Forward prime | 2 μl |
| Reverse primer | 2 μl |
| Template | 2 μl |
| Enzyme (mix) | 44 μl |
Because there are air bubbles in the tube, perform centrifugal operation. Put it into the PCR machine and set the program.
Experimental report 7.21.2021
(1)Enzyme Digestion
Restriction endonucleases each microliters (double digestion Ndel, Xbal)
| Total | 50 μl |
|---|---|
| buffer | 5 μl |
| plasmid | 5 μl |
| Ndel | 3 μl |
| Xbal | 3 μl |
| Distilled water | 34 μl |
(2)Electrophoresis
① made agarose gel② droped the vector, fragment, and marker respectively (control, point on the outermost side)③ After 20 minutes, the agarose gel was irradiated with ultraviolet light, and bright bands were visible.Notice, the control marker can verify the bp of the vector and fragment.
(3)Recover gene fragments
①. Added 250 μl of Buffer BL to the adsorption column EC, centrifuge at 12,000g for 1 min to activate the silica gel membrane; ②. Under a 365nm long-wave UV lamp, used a clean blade to cut the DNA strips that need to be recovered, and try to cut off the DNA-free gel, and the smaller the gel volume, the better ③. Put the gel containing the target DNA band into a 2mL centrifuge tube; D. Add 500μL of Buffer GL; ④. Water bath at 65°C for 4~6min, and mix up and down every 2~3min until the gel is completely melted and the solution is light yellow (if the gel mass is too large, add Buffer GL to the solution until the solution is light yellow) This time it’s been heated for about 13 minutes because the rubber is too big. ⑤. Transfered the solution to the adsorption column EC, centrifuge at 12,000g for 1 min, discard the waste liquid, and put the adsorption column EC back into the empty collection tube; ⑥. Added 700 uL of Buffer W2 to the adsorption column EC (please check whether the specified volume of absolute ethanol has been added, centrifuge at 12,000g for 1 min, and discard the waste; ⑦. Repeated step 7 once; ⑧. Put the adsorption column EC back into the empty collection tube and centrifuge at 12,000g for 2 min; ⑨. Took out the adsorption column EC, put it in a clean 1.5mL centrifuge tube, open the lid at 20-25 and let it stand for 2 minutes, and add 35-50 uL Eluent to the middle of the adsorption membrane (60-65°C preheating the Eluent is better) ⑩ Leaved it at 20~25℃ for 2min, and centrifuge at 12,000g for 2mi. If a larger amount of DNA is required, the obtained solution can be transferred to the adsorption column again and centrifuged for 2 minutes The larger the elution volume, the higher the elution yield. If a higher concentration of DNA is required, the elution volume can be appropriately reduced, but the minimum volume should not be less than 25 μL. If the volume is too small, the elution yield will be reduced.
Experimental note 7.25.2021
(1) Measure DNA content.
(2) Knowing how to use Spectrophotometer.
Since DNA is in Eluent, eluent zero-adjusting spectrophotometer is required.
The first zero adjustment test: 4.5.
The second zero adjustment test: 1.5.
Putting in the fragment for the first time: -88.5(large error).
The third zero adjustment test: -71.5.
The fourth zero adjustment test: -1.
Put in the carrier: 25.5.
Put in the fragment: 4.5.
Experimental note 7.26.2021
Plasmid extraction
① Preparations.
Check if RNase has been added to Buffer1.
Check whether ethanol has been added to Wash Solution.
Check for precipitation in Buffer2 and P3.
②Take 1.5-5ml of the bacterial solution cultured overnight, centrifuge at 8,000rpm for 2 minutes to collect the bacterial cells, and discard the culture medium.
③ Add 250 ul Buffer P11 to the sediment to completely suspend the bacteria.
④ Add 250 ul Buffer P22, and immediately mix by gently inverting the centrifuge tube 5-10 times. Let stand at room temperature for 2-4 minutes. ⑤ Add 3500 ul Buffer P33, and immediately mix by gently inverting the centrifuge tube 5-10 times. ⑥Centrifuge at 6.12,000×g for 5-10 minutes. Transfer the supernatant to the adsorption column, centrifuge at 8,000×g for 30 seconds, and discard the liquid in the collection tube. ⑦ (Optional) Add 500 l Buffer DW11, centrifuge at 9,000×g for 30 seconds, and discard the liquid in the collection tube. ⑧ Add 500 ul Wash Solution, centrifuge at 9,000×g for 30 seconds, and discard the liquid in the collection tube. ⑨ Repeat step 8 once. ⑩ Centrifuge the empty adsorption column at 9,000×g for 1 minute. ⑪ Put the adsorption column into a clean 1.5ml centrifuge tube, add 50-100 ul Elution Buffer to the center of the adsorption membrane, leave it at room temperature for 1 minute, and centrifuge for 1 minute. DNA solution in storage tube.
Experimental note 7.27.2021
(1) Measure the DNA concentration.
Results: the concentration of digested nucleic acid is 540 ng/μL and 308 ng/μL. The concentration of GFP fragment is 525 ng/μL and 168 ng/μL
(2) Agarose gel electrophoresis.
Configuration: 1% agarose solution plus 2 microliters of nucleic acid staining solution.
Experimental report 7.28.2021
Homologous recombination.
10 μl system Enzyme: Fragment: Vector=5:4:1.
Actual operation:
| Total | 10 μl |
|---|---|
| Carrier | 2.5 μl |
| Fragment | 2.5 μl |
| Enzyme | 5 μl |
Put it in the PCR machine at 50°C for 1h, and store at 16°C.
Experimental report 7.29.2021
Plasmid transformed Escherichia coli DH5α
(1) Mixed 50 μL E.coli DH5α and 5 μL vector - target gene recombination produst gently and held on ice for 30min.
(2) In 42℃ water bath heat shock bacteria 90 s, and qucinkly put the mix into the wet ice. Let it stand for 2 min.
(3) Added 1 mL liquid LB medium into the mix and incubate it at 37℃ constant temperature box table, 220 rpm shaking the culture for 1h.
(4) Centrifuged the culture at 5000 rpm for 2 min and discard the supernatant.
(5) Resuspended the bacterial residue with 100 μL sterile LB and spread on LB plate containing apramycin, then incubate overnight at 37℃ for 10-12 h.
Selected the positive clones by using colony PCR.Experimental note 8.1.2021 Colony PCR.
| Total | 10 μl |
|---|---|
| 2x mix enzyme | 5 μl |
| Front primers | 0.5 μl |
| water | 4 μl |
| Back primers | 0.5 μl |
PCR machine program:
Experimental note 8.2.2021
Electroconversion
①Dissolved competent cells for electrotransformation on ice at -80℃. ②Added appropriate amount of pre-chilled DNA fragments and mix well. ③Transfered to the pre-cooled electro-rotor cup in batches. ④Took the appropriate voltage and resistance to convert with the electroconverter 2500V voltage 4.5-5.5ms. ⑤Quickly added 500 microliters of LB (pre-cooled) to the electro-rotor, mix well, transfer to a sterile centrifuge tube, and recover at 37°C for 1 hour. ⑥Centrifuged at 5000 rpm for 1 min, remove most of the supernatant, and resuspend the bacteria with the remaining supernatant. The bacteria liquid is mixed and placed on an LB plate containing three antibiotics, 37 degrees for 12-16h.
Finally, colony PCR verification.
Experimental note 8.5.2021
ET processing:
Suck bacteria, centrifuge, discarded waste.
Saved with sterile LB.
Spore treatment:
Placed the spores in water, add glass beads to disperse, shake to suspend the spores in the water, and store with 15% glycerin.
Spores 50°C water bath heated shock for 10min.
Mix spores and ET, and coat MS plate:
Magnesium ions can help spore germination
① MS tablet formula 2g soybean powder, add 100mL distilled water, boil and filter, dilute to 100mL ② Spore pre-germination medium 1% casamino acid, 1% yeast extract, after sterilization, add calcium chloride to a final concentration of 10mM ③Apramycin (50mg/ml): dissolve in distilled water ④ Nalidixic acid (25mg/ml): 0.15mol/l NaOH dissolved ⑤ Kanamycin (50mg/ml): dissolved in distilled water ⑥Chloramphenicol (25mg/ml): dissolved in absolute ethanol ⑦TES (0.05M): 0.573g/50ml, adjusted to pH 8.0 with NaOH
Use a one-ml pipette in the ultra-clean workbench to draw the pre-prepared actinomycete seed solution, and evenly pour it into the sterilized feather culture medium, and quickly cover with the sealing film (in advance, put the bottle mouth in the alcohol lamp Sterilized on flame)
Experimental note 8.7.2021
(1) Covered the MS plate with 1 mL of sterile water containing apramycin and nalidixic acid, and blow-dry it on a clean bench
(2) BCA protein detection method
①Used different concentrations of calf serum protein to make a standard curve (the relationship between absorbance and concentration)
② 25μl sample + 200μl working solution
Working fluid A: B=100:1
Incubated at 37°C for 30min,
Measured the absorbance at 562nm and compare the curve
Experimental note 8.10
Picked the positive zygote and culture it into LB with apramycin and nalidixic acid.
Experimental note 8.11
Inoculate the bacterial liquid with an absorbance of 9 at 600nm into 10% feather culture medium, with an inoculum amount of 1%.
Experimental note 8.12
Sampling after 24h fermentation.
Experimental note 8.13
Took samples after 48 hours of fermentation.
Experimental note 8.14
Measured the amino acid and peptide content and keratinase activity of 24h samples and 48h samples.
Experimental note 8.15
Measured the 48h degradation rate.