Team:NJMU-China/Protocol
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Perparation
LB medium
Liquid(1L):
| Tryptone | 10g |
| NaCl | 10g |
| Yeast | 5g |
Solid(1L):
| Tryptone | 10g |
| NaCl | 10g |
| Yeast | 5g |
| Agar | 15g |
5-HT : Serotonin 5.2863 mg + dd water 30 mL
HSL : 1.1 mg + DMSO 3 mL
X-Gal ：20 mg + DMSO 1 mL
—— written by Linfeng Zhao
IPTG induction on agar plates
1. Mix the appropriate amounts of chromogenic substrate X-Gal and the inducer IPTG, then spread on agar plates containing antibiotics.
2. Incubate the plates in an obverse position for 4 hours at 37℃ and wait for drying
3. Spread the overnight Cultured Bac on the plate containing X-Gal and IPTG. Incubate the plate overnight at 37℃.
4. Add 5-HT diluent on the surface of agar plates in the experimental group, meanwhile, add HSL diluent in the control group.
5. Wipe any condensation from the lid of the plates. Then incubate the plates in an inverted position at 37℃, to allow the blue color to develop.
—— written by Bujie Xu
Plasmid extraction
1. Resuspend the pelleted bacterial culture in 250 μL of the Resuspension Solution (RNase A has been added to the Resuspension Solution). Transfer the cell suspension to a microcentrifuge tube. The bacteria were resuspended completely by vortexing until no cell clumps remain
2. Add 250 μL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
3. Add 350 μL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. Centrifuge for 10 min to pellet cell debris and chromosomal DNA.
4. Transfer the supernatant to the supplied spin column by pipetting. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
5. Add 600 μL of the Wash Solution (diluted with ethanol) to the spin column. Centrifuge for 30 seconds and discard the flow-through. Place the column back into the same collection tube.
6. Repeat the wash procedure using 600 μL of the Wash Solution.
7. Discard the flow-through and centrifuge for an additional 2 min to remove residual Wash Solution.
8. Transfer the spin column into a fresh 1.5 mL microcentrifuge tube. Add 100 μL of the Elution Buffer to the center of spin column membrane to elute the plasmid DNA. Incubate for 2 min at room temperature and centrifuge for 2 min.
9. Discard the column and store the purified plasmid DNA at -20℃.
—— written by Bujie Xu
Protein extraction
1. Preparation of extract
According to the required sample volume, add 2ul protease inhibitor and 5ul protein stabilizing solution to every 500ul extract A, mix well and place on ice for later use.
2. Centrifuge the bacterial solution at 10000xg for 5 minutes at 4℃, discard the supernatant, try to absorb the remaining liquid, and collect the bacteria.
3. Wash the bacteria twice with PBS. If the bacteria are frozen, proceed directly to the following steps.
4. Add 500ul protein extract for every 100mg wet weight of bacterial sample, mix by pipetting, and place on ice for 20-30 minutes.
5. Under the conditions of 150-300w, 10s ultrasound/10s interval, ice bath ultrasound until the bacteria liquid becomes clear.
6. Centrifuge the extract for 5 minutes at 4℃ and 12000xg, discard the precipitate, and collect the supernatant.
7. Transfer the supernatant to another clean centrifuge tube to obtain a Gram-positive bacterial protein sample.
8. Quantify the total protein sample and place it in a refrigerator at -80℃ for later use or directly use in the next experiment.
—— written by Linfeng Zhao
Protein concentration measurement
1. Dilute BSA standard substance: use diluent consistent with protein sample which is under test to dilute BSA standard
| Name | Amount of diluent (ul) | Amount of BSA standard substance (ul) | Final concentration of BSA standard (ug/ul) |
|---|---|---|---|
| A | 100 | 100 | 1 |
| B | 100 | 100(take from A) | 0.5 |
| C | 100 | 100(take from B) | 0.25 |
| D | 100 | 100(take from C) | 0.125 |
| E | 100 | 100(take from D) | 0.0625 |
| F | 100 | 100(take from E) | 0.03125 |
| G | 100 | 0 | 0 |
2. Preparation of BCA working liquid:
a. calculate the BCA working liquid amount:
total BCA working liquid = (BSA standard sample number + unknown sample number) × hole number × volume of each BCA working liquid
b. according to calculate the amount of BCA working liquid, mix the BCA - A Solution and BCA – B Solution according to the volume ratio of 50:1 to prepare BCA working liquid, blending in full
3. Quantitative detection:
1) according to the table, add 25ul diluted A – G BSA standard substance and protein samples under test to every 96 hole microplate which is tagged respectively, recommend each sample under test for 2-3 parallel reactions. (according to the concentration of the sample, dilute 5 or 10 times ahead)
2) add 200 ul BCA working liquid to each hole, blending in full, cover the 96 hole microplate, 37℃ incubation for 30 minutes, cool to room temperature, complete the test within 3 to 5 minutes.
3) use spectrophotometer or instrument of enzyme to determination the absorbance value of each sample and BSA standard at 562 nm, record at the same time.
4) draw standard curve, calculate the protein concentration in the samples
—— written by Sijing Shi
SDS-PAGE
A. Preparation of reagents
1.10 x electrophoresis buffer (pH 8.3) : Tris 3.02g, glycine 18.8g, dissolve 10% SDS 10 ml with ddH20 , the capacity to 100 ml.
2.coomassie brilliant blue staining fluid: coomassie brilliant blue 0.25g;methanol 225 ml, acetic acid glacial 46 ml, ddH20 225ml .
3. decoloring liquid: methanol 300ml, acetic acid glacial 100ml, ddH20 600ml.
B. Operating steps
Using vertical electrophoresis tank device
1) The configuration of the separating gel (10%) :
| ddH2O | 4.0ml |
| 30% reserves acrylamide | 3.3ml |
| Tris-HCL | 2.5ml |
| 10% SDS | 0.1ml |
| 10% AP | 0.1ml |
2) The configuration of the laminated gel (4%)
| ddH2O | 1.4ml |
| 30% reserves acrylamide | 0.33ml |
| Tris-HCL | 0.25ml |
| 10% SDS | 0.02ml |
| 10% AP | 0.02ml |
| TEMED | 2ul |
Remove the water on the separating gel, add the mixture, insert the comb between glass immediately, completely polymerization needs 15 to 30 min.
Take 1 ml of the above mixture, add TEMED 4 ul, blend and then pour into the glass. Add water on the top, pay attention to make the surface flat, (gel completely polymerization needs 30 to 60 min).
1. The configuration of the polypropylene acyl gel.
2. Sample treatment: join the sample in the same amount of 2X SDS buffer, 100℃ heat for 5 min, centrifugal 12000 g x 1min, take supernatant for SDS-PAGE analysis, at the same time the SDS standard low molecular weight protein for parallel processing.
3. take 10 ul induction and its processed samples to join sample pool, and add 20 ul low molecular weight protein standard as contrast.
4. add 1 x electrophoresis buffer in electrophoresis tank, connect the power supply, the voltage for separating gel is 60V, the voltage for laminated gel is 100V, stop electrophoresis when the bromophenol blue reach the electrophoresis tank bottom (about 3 hr).
5. Staining: remove gel from the glass plate, coomassie brilliant blue staining fluid dyeing, 4-6 hr at room temperature.
6. Decoloring: remove gel from dyeing liquid, put in bleaching liquid, multiple decoloring until the protein is clear.
7. Take a picture and save.
—— written by Sijing Shi
Expression of protein in E.coli by IPTG induction
1. Inoculate 50 ml of LB Medium with 5 ml of an overnight cultured bacterial suspension in the conical flask. Incubate at 37ºC with vigorous agitation. Grow the cells to log phase (OD650 0.5-0.8).
2. Add 240 ul of the inducer IPTG (50mg/ml) to the induced group.
3. Continue Incubating at 37ºC.
4. Collect the bacterial solution after 2 hours, 4 hours, and 6 hours.
5. Extract protein from the obtained bacterial solution, then store at -80℃.
—— written by Bujie Xu
The count of E. coli
1. The cultivation of the bacteria：
Add 10ml LB, 10ul ampicillin, 10ul Glycerin bacteria to 15ml centrifuge tube, cultivate in 37℃ for 14-16h.
2. Diluting and coating:
| Name | Glycerin bacteria(ul) | ddH2O(ul) |
|---|---|---|
| A | 40 | 360 |
| B | 40(take from A) | 360 |
| C | 40(take from B) | 360 |
| D | 40(take from C) | 360 |
| E | 40(take from D) | 360 |
add 50ul A – E sample to solid LB, spread in full, cultivate in 37℃ for 18h.
3. Count the colonies on the plate and record.
—— written by Sijing Shi