Team:AHUT China/protocol

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   Protocols

    

LB Medium（1L）：

 

 tryptone

10g

yeast extract

5g

NaCl

10g

ddH2O

 

Adjustable pH: 1L plus 200μl,5mol/L NaOH pH to 7.0





LB solid Medium（1L）：

 

 tryptone

10g

yeast extract

5g

NaCl

10g

agar

15g

ddH2O

 

Adjustable pH: 1L plus 200μl,5mol/L NaOH pH to 7.0




 

50*TAE（1L）

 

ddH2O

500ml

Tris buffer solution

242g

0.5mol/Ll-1 EDTA

100ml

Glacial acetic acid

57.1ml

ddH2O

 

 

0.25 %  dye(1L)

 

G-250

100mg

90%ethanol

50ml

85% phosphoric acid

10ml

ddH2O

 

 

10 % separation gel(10ml)

 

distilled water

1.9ml

30%Arc-Bis

1.7ml

1.5M Tris-HCl pH 8.8

1.3ml

10%SDS

0.05ml

10 % ammonium persulfate

0.05ml

TEMED

0.002ml

 

5 % concentrate glue(6ml)

 

distilled water

2.7ml

30%Arc-Bis

0.67ml

1.0M Tris-HCl pH 6.8

0.5ml

10%SDS

0.04ml

10 % ammonium persulfate

0.04ml

TEMED

0.004ml

 Nickel column purification buffer
① 5×imidazole 500ml（pH7.5）
50mM Tris       3.0285g
100mM NaCl       2.922g
5mM Imidazole       0.1702g
② 50×imidazole 500ml（pH7.5）
50mM Tris       3.0285g
100mM NaCl       2.922g
50mM Imidazole       1.702g
③ 250×imidazole 500ml（pH7.5）
50mM Tris       6.057g
100mM NaCl       5.844g
250mM Imidazole       8.51g
④Desalination 500ml（pH7.5）
10mM Tris       0.6058g
100mM NaCl       2.922g

 

Enzyme buffer solution:

 

buffer solution

1ml   990 μL   980 μL  970 μL   960 μL

enzyme（1 μg/mL）

0μL  10μL  20μL  30μL  40μL

Bromothymol blue indicator

20μL  20μL  20μL  20μL  20μL

 

P-nitrophenol

 

concentration

0.01 mM   0.02 mM   0.03 mM  0.04 mM   0.05 mM

p-NP（0.1 mM）

2 mL  4 mL  6 mL  8 mL  10 mL

ddH2O

18 mL  16 mL  14 mL  12 mL  10 mL

 

Gel Extraction:

Preparatory work:

1. Before the first use, Buffer W2 concentrate was added to the specified volume of anhydrous alcohol.

2. Prepare Tip heads and centrifuge tubes without nucleic acid and nuclease contamination.

3. Prepare 75C water bath.

4. Check whether Buffer DE-B precipitates before use. If precipitates occur, they should be melted in a 70 degree C bath and cooled to room temperature before use.


Action step:

1. An agarose gel containing the intended DNA is cut under a UV lamp to calculate the gel weight, which acts as a 1 gel product (e.g., mg=100μl volume).

2. Add 3 Gel Product buffer DE-A, mix evenly after 75°c heating, intermittent mixing (per 2-3 min), until the gel block completely melted (about 6-8 min).

3. Add 0.5 buffer DE-B Volume buffer DE-B, mix evenly. When the isolated DNA fragment is less than 400bp, it is necessary to add another 1 gel product of Isopropyl Alcohol. Add Bufer DE-B After the mixture color changed to yellow, fully mixed to ensure the formation of a yellow solution.

4. Take the mixture from step 3 and transfer to the DNA preparation tube (provided in the kit) in a 2 ml centrifuge tube, 12,000x g 1min. Discard the Filtrate.

5. Put the preparation tube back into 2ml centrifuge tube, add 500μl buifer w1,12,000xg centrifuge 30s, Discard Filtrate.

6. Place the preparation tube back into 2 ml centrifuge tube, add 700μl Buffer w2,12,000xg centrifuge 30s, Discard Filtrate. In the same way again with 700μl Buffer W2 Wash once 12,000xg centrifuge 1 min. Confirm that anhydrous ethanol has been added to the Bufer W2 concentrate by the specified volume on the reagent Bottle. Two plays using buffer W2 flushing ensures that the salt is completely removed, eliminating the effects on subsequent Experiments.

7. Place the preparation tube back into the 2 ml centrifuge tube, 12,000xg Centrifuge 1 min.

8. Place The tubes in a clean 1.5ml centrifuge tube, add 25-30μl eluent or deionized water at the center of the preparation film, and 1min at room Temperature. 12,000xg Centrifuge 1 min to elute DNA.


Pick Monoclonal:

1.Take 14ml lb liquid culture liquid in 50ml centrifuge tube.
2.Add 14μl ampicillin.
3.Use the small gun head to pick a single colony and throw the head into the 50ml centrifuge tube.
4.37℃,200rpm Shaker Culture Overnight.


Preparation of competent cells:

1.Pick a single colony inoculation to 2.5ml lb liquid medium (15ml centrifuge tube), loosen about 1/3, 37℃,250rpm, Shaker culture overnight.
2.Take 300μl bacterium liquid to add 15mlLB medium of 50ml centrifuge tube, culture 5-6 hours, take 100μl bacterium liquid, measure OD600 Value.
3.The above 50ml centrifugal tube ice bath 30min. The remaining bacteria liquid in the centrifuge tube is divided into several centrifugal tubes,
4.4℃,4000rpm centrifugal 5min, to the Supernatant.
5.With 6ml pre-cooled CaCl2 resuspend strain, ice bath 30min.
6.4℃,4000rpm Centrifuge 5min, go to the Supernatant.
7.Resuspend the bacterial solution with 0.6 ml of CaCl2 containing 10% glycerol，Dispense in 100 μl tubes on ice..


Miniprep:

1. Take 2.5ml in lb medium to cultivate overnight bacterial solution (if using rich medium, the liquid product should be halved or less), 12000*g, centrifuge 1min, Discard the Supernatant.
2. Add 250μl Buffer S1 suspended bacteria precipitate, the suspension need to be uniform, there should be no small bacteria block.
3. Add 250μlbuffer S2, gently and completely upside down 4-6 times mix evenly make the bacteria full lysis, until the formation of a translucent solution, this step should not exceed 5min.
4. Add 350μlbuffer S3, gently and fully upside down mix 6-8 times, 12000*g Centrifuge 10min.

Centrifugal method:

5. Take the centrifugal supernatant in step 4 and transfer to the preparation tube (placed in a 2ml centrifuge tube (provided in the kit)), 12000*g centrifuge 10min, Discard the Filtrate.
6. Put the preparation tube back into the centrifuge tube, add 500μlbuffer w1,12000*g Centrifuge 1min, Discard the Filtrate.
7. Put the preparation tube back into the centrifuge tube, add 700μlbuffer w2,12000*g Centrifuge 1min, Discard the filtrate; in the same way again with 700μlbuffer W2 wash once. Discard the Filtrate
8. Place the preparation tube back into the 2ml centrifuge tube, 12000*g Centrifuge 1min.
9. Move the preparation tube into the new 1.5ml centrifuge tube, add 60-80μleluent or deionized water at the center of the preparation tube, and 2min at room Temperature. 12000*g Centrifuge 1min. 10.-20℃ Save.


1% Identification of gel:

1.Take 0.2g agarose.
2.Add 20ml 1*TAE Solution.
3.In the conical bottle dissolved, mixed, sealed with breathable sealing film, in the microwave oven began to heat 2 minutes, the bottle in the bubble immediately stop heating, with a towel to the cone bottle out, Shake well, Repeat steps until the solution becomes clear.
4.When the bottom of the bottle is not hot when adding 2μl nucleic acid dye (with The solution ratio 1:10,000), pour into the cleaning of the small tooth comb in the rubber plate, after the gel solidified, removed, placed in the electropHoresis pond, prepared on the sample, electrophoretic liquids submerging gel surface
5.Mix the sample with the upper sample dye at a ratio of 10:1. Place the sample into a hole and place the 2μl molecular weight marker into the first hole.
6.At the end of electropHoresis, 302nm light glue was used in Ultraviolet analyzer to record the experimental results.


1% Recycling used gel:

1.Take 0.3g agarose.
2.Add 30ml 1*TAE Solution.
3.In the conical bottle dissolved, mixed, sealed with breathable sealing film, in the microwave oven began to heat 2 minutes, the bottle in the bubble immediately stop heating, with a towel to the cone bottle out, Shake well, Repeat steps until the solution becomes clear.
4.When the bottom of the bottle is not hot when adding 2μl nucleic acid dye (with The solution ratio 1:10,000), pour into the cleaning of the small tooth comb in the rubber plate, after the gel solidified, removed, placed in the electropHoresis pond, prepared on the sample, electropHoresis liquid has not been rubber surface.
5.Mix the sample with the upper sample dye at a ratio of 10:1. Place the sample into a hole and place the 2μl molecular weight marker into the first hole.
6.At the end of electropHoresis, 302nm light glue was used in Ultraviolet analyzer to record the experimental results.


Transformation:

1.Take the plasmid containing the 1μl DNA and add it to the 1.5ml EP tube containing the 50μl competent cells, mix with the gun head, ice bath 30min.
2.Move the EP tube into the 42 ℃ water bath and heat shock 90s.
3.Quickly move the EP tube into the ice bath in 2min.
4.Added 0.4ml non-resistant liquid lb medium (at room temperature), 37 ℃ Shaker to cultivate 45min.
5.Apply the bacterial solution to the plate containing the antibiotic, positive 10min.
6.Placed upside down in a 37 ℃ incubator and cultured for 12-16 Hours.


Purification of Proteins:

1.With IPTG as inducer induction strain enlargement: 1:100 of the percentage of lb medium (kanamycin concentration of 50μg/ml), that is, add 2ml of bacteria, placed on the shaker, 37℃ 200rpm culture, When the OD600 value reaches 0.6〜0.8, take 4ml bacteria to separate culture control, and mark, the remaining added IPTG, the final concentration of 0.5mM, placed on the shaker, 37 ℃ to 〜200rpm Culture 4 hours, and then take 4ml after the induction of the bacteria, two of the bacterial liquid extractedby the previous steps to extract the protein, and Sds-page identification, after the identification, the remaining bacteria to 4 ℃, 12000rpm Centrifuge 10 minutes, to the supernatant, the bacteria remain in the -20℃ Refrigerator.

2. Samples were treated by centrifugation of iptg-induced bacteria, thawed at low temperature, and added about 30mL cell lysis fluid to resuspend the Strain. The probe of ultrasonic cell crusher was cleaned with 20% ethanol and distilled water, and the probe was inserted into the centrifuge tube, which was fixed in the ice, so that the probe could not reach the centrifugal tube wall. Start the ultrasonic cell crusher, set the power to 200W, work 20min. If the crushing effect is not ideal, can be broken again 20min. Close the ultrasonic cell crusher and wash the probe with 20% ethanol and distilled water for the next use. The broken bacteria liquid 9000rpm centrifuge 30min, take 1mL supernatant and the right amount of precipitation to mark and put into the Refrigerator.

3. Buffer Solution preparation: preparation of balanced buffer (5x imidazole), elution buffer (50x imidazole, 250x imidazole) and desalination buffer

4. Purification using Ni-nta-μspHere (5ml) pre-assembled nickel column for purification, operating environment in the 4-degree chromatograpHy cabinet, Imidazole and Desalting are pre-cooled at 4 degrees in Advance. Approximately 30 1.5ml EP tubes are prepared and add 1ml G250 to each tube for inspection during Purification. The following steps are as Follows:
1) Assemble the ni-nta-μspHere kit, clean the tube outside the peristaltic pump pipe, Insert the pipe into the purified water, and start the peristaltic pump to clean the inside of the Pipe.
2) Insert the pipe into the 5x imidazole solution of the ice bath, start the peristaltic pump, run the pipe and nickel column.
3) Use the supernatant after centrifugation, the peristaltic pump speed is slow, the speed is 5rpm.
4) Start the peristaltic pump, the pipeline into the ice bath 50x imidazole solution, to wash away the miscellaneous protein, the speed of 7rpm, the outflow liquid preparation sample Preservation. Every 2min with test Coomassie Blue G-250 solution to test the effluent, if the solution does not change color, can be considered as impurity protein has been removed.
5) At this time to change into the ice bath 250x imidazole solution to wash away the purpose of the protein, the speed of 7rpm, every 2min with test Coomassie Blue G-250solution Test effluent, If the solution discoloration, proved that the purpose of the protein is washed off, at this time with an ice water bath in the concentrated tube (according to the size of the protein determined ...). Receiving outflow fluid. If there is no protein outflow in every 2min, if the protein is out, then clean the pipe and nickel column into pure water.
6) Centrifuge the concentrate tube in a 4 500 rpm centrifuge for 10 minutes, and take out the concentrate tube and cool it in ice water for about 5 minutes. Repeat the above steps until the volume of solution in the concentration pipe is less than 2mL (this process takes about 6h time).
7) Add 2mL desalting solution to continue centrifugation (this process takes about 3H time). When the solution in the concentrate tube is about 1000 uL, the solution is carefully sucked out by a pipette in a small centrifugal tube, marked and stored at - 4 C. The remaining solution was packed into five sterilized tubes. 100 ml solution was added with 100% glycerol (glycerol was thick enough to be sucked at the end of the gun and mixed with the gun blowing, no oscillator was used). The solution was frozen and stored in a - 80 degree refrigerator.
7) Preservation of chromatographic column: For long-term storage, the column can be washed with distilled water, then cleaned with 20% ethanol and stored in 20% ethanol solution. If only overnight storage is needed, the resin buffer can be stored in the chromatography column.
8) Regeneration of chromatographic columns: if chromatographic columns have been used, chromatographic columns should be regenerated before experiments. The washing sequence is: ddH2O 8mL 0.1M EDTA 8mL ddH2O 8mL 0.5M NaOH 2ml soaking for 30 minutes ddH2O 20mL 0.1M NiSO4 8mL ddH2O 8mL.
If the chromatographic column can be used directly on the day, it should be kept in 20% ethanol for the day.


Expression and identification of protein

1.Expression of protein

1) In the Ultra-clean table, the enlarged cultured bacteria were added to the two 4 ML lb medium (kanamycin concentration of 50ΜG/ML) by 1:100, adding 40μl bacteria;
2) Placed on the shaker, 37 ℃, 200 rpm for 4 hours. culture;
3) When the OD600 value reaches 0.6〜0.8, one of the two tubes joins the iptg, and the final concentration is 0.5 mM;
4) On the shaker, 37 ℃, culture for 4 hours.

2. Protein extraction
1) Whole-cell lysate: respectively, from the two tubes of bacteria to absorb 200μl bacteria liquid, in the refrigerated centrifugewith 4 ℃, 12000 rpm Centrifuge 10 minutes, to the supernatant, and 100μl 1x SDS Loading buffer re-suspended precipitation, and then in 100 ℃ water bath pot added Heat for 10 minutes and put in the -20℃ refrigerator to Save.
2) Supernatant and precipitation of cell lysate: the remaining bacteria in the two tube is divided into labeled 1.5 ML EP tube, in the refrigerated centrifuge with 4 ℃, 12000 rpm Centrifuge 10 minutes, go to the supernatant, collect the bacteria, with 1 ML PBS wash precipitation, and will mark the same in the same EP tube of bacteria suspended together. Centrifuge in the freezer 4 ℃, 12000 rpm centrifugation for 10 minutes, to the supernatant, add 3 ML of cell lysate, resuspend the bacteria, with the ultrasonic crusher broken bacteria, The procedure is as Follows: 200w, 3s interval, ultrasonic 20 minutes, make the bacteria clear. Extract the 300μl bacteria solution from the ultrasonic bacteria, centrifuge for 10 minutes at 4 ℃, 12000 rpm, and use 1.5 ML EP tube to collect the supernatant and precipitate of cell lysis, and Mark Well. Add the 70μl 5x SDS Loading Buffer resuspension to the supernatant, add 300μl 1x SDS Loading Buffer resuspension to the precipitate, then heat 10 minutes in a 100 ℃ water bath and put in a -20℃ refrigerator.

3. Identification of protein
1) Determination of gel concentration;
2) Preparation of separation gel;
3) Install the glass plate and fix it properly. Quickly inject the separation glue between the two glass panes until the remaining plate width is 1 cm more than the length of the comb, and then add 1 ML of anhydrous ethanol on the separation surface; 
4) After the separation gum polymerization complete, pour the anhydrous ethanol, and repeatedly rinse the adhesive surface with distilled water several times, and then use the filter paper to suck the residue on the adhesive surface;
5) Prepare the concentrated glue: insert the clean comb immediately after pouring the concentrated glue, do not produce bubble;
6) The gel into the electropHoresis tank, and installed, in the upper and lower electropHoresis tank to add enough electropHoresis buffer;
7) On the sample, the sample order is as Follows:

First lane: 2μlmarker;
Second lane: 30μl total protein without iptg;
Third lane: total protein of 30μl plus iptg;
The four lanes: 30μl without iptg;
The five lanes: 30μl not add IPTG precipitation;
The six lanes: 30μl plus Iptg's supernatant;
Seventh lane: precipitation of 30μl plus iptg.
8) correct access to the positive and negative electrodes, first with 80V voltage electropHoresis, The sample is condensed into a thin line, to increase the voltage to the isolation sample, when the strip ran to the end of the gel, stop electrophoresis;
9) Remove the plastic block, the electropHoresis of good gel into the appropriate amount of G250 test dye solution, Shaker room temperature staining for 30 minutes;
10) dye good glue into the decolorization of the liquid to be discolored, when the decolorization of blue to replace the decolorization liquid, until the background is colorless and the protein bands Clearer.




Sds-page and Western blot identification of proteins

1. Transfer film
1) Preparation of electropHoresis gels for sds-page;
2) After electropHoresis, the tape is cut to the appropriate size, with the transfer buffer (pre-cooling) balance;
3) Pre-cut the same size as the tape filter paper and NC membrane, immersed in the film buffer of ten min;
4) The transfer film device from bottom to top, according to the anode Carbon plate, filter paper, NC film, gel, filter paper, Cathode Carbon platesequence, filter paper, gel, NC film Accurate alignment, each step to remove the bubble, the carbon plate on the excess liquid absorption dry;
5) Switch on the power supply, set the voltage 100V, transfer 1.5 hours, disconnect the film out, cut the film to be tested for WB.

2. Staining and antibody incubation
1) Dyeing with Lai Chun red dye for a period of time after removal, adding tbst for decolorization;
2) The good color of the NC film into the new milk to be closed, after 1 hours, adding a resistance, incubate for 2 hours;
3) After the first anti-incubation, with TBST several times to clean, then add two resistance, incubate for 1 hours, the end of incubation, with TBST Cleaning.

3. Development
1) Using a luminescent detector to incubate the NC membrane for a period of time, the NC membrane transferred to the pHotograpHic cassette, and then the box Zhong Pu on the cling film, the film bubble discharge;
2) Pour the prepared developer and fixer into the tray, place the tray in the developing room and turn on the infrared lamp;
3) Take the film box into the darkroom, pay attention to avoid light, then cut the appropriate film in the darkroom, and do a good mark, the film is covered on the NC membrane, close the pHotograpHic cassette, wait a few seconds, take out the film, put into the development of the development liquid, and then after the strip shows, wash with water, and then put in the
4) When finished, dry the film, Mark it, andtake a picture.

4. Quantification of protein concentrations
1) Preparation of 0, 0.125, 0.5, 0.75, 1, 1.5 mg/ml protein standard solution;
2) the standard solution of different concentrations of 10μl was added to the 96-well plate;
3) Take 10μl sample into the sample hole of the 96-well plate;
4) Add 300μl G250 staining agent to each hole;
5) Detection with an enzyme mark, 630 nm to determine absorbance;
6) The standard curve is made according to the absorbance of the protein standard solution; 
7) The protein concentration in the sample is calculated according to the standard curve and the sample Volume.


Determination of enzymatic activity of carbonic anhydride

1. Colorimetric assay enzyme Activity
1) Preparation of bromine thyme Blue indicator, the 0.10 g bromine thyme blue soluble in 8.0 ml 0.02 mol/l sodium hydroxide solution, diluted to 250 ml (yellow 6.0--7.6 blue);
2) The preparation of Tris-hcl buffer, said to take 3.0285gTris placed in a beaker, adding 800mL to dissolve water mixing, adding hydrochloric acid to adjust pH to 8.3, transferred to the capacity of the bottle, plus shuiding, high temperature sterilization after the temperature reserve;
3) The CO2 will be bubbling to about 3 hours in distilled water, preparing CO2 saturated solution, and then cooling the CO2 solution in an ice bath;
4) Preparation of 1mL different concentrations of the enzyme buffer solution
5) In the 1.5 ML EP tube added to the above-mentioned enzyme buffer, and then quickly joined 250μl CO2 saturated solution, began to use a stopwatch to record the amount of time the liquid changed from blue to YELLOW. The same concentration of enzyme buffer to do three times, the error to control within 1 s;
6) Two test tubes were added to 1mL newly prepared 3mmp-npa, a 0.1 m PBS buffer with 0.3 ml enzyme (1ΜG/ML) and 1.7 ml, and a 2 ml 0.1 m PBS buffer solution with a UV spectropHotometer to measure absorbance values at the wavelength of 400nm Repeat 3 times;
7) After 3 min, the absorbance value is measured by ultraviolet spectropHotometer at the wavelength of 400 nm;
8) Compare the standard curve and calculate the conversion Rate. Enzyme activity is defined as: at room temperature, hydrolysis of 1μmol acetate per minute of the amount of enzyme required for nitrobenzene ester is an enzyme unit.


Quantification of protein concentration


1) Prepare 0, 0.125, 0.5, 0.75, 1 and 1.5 mg/mL protein standard solutions.
2)10 mg L of different concentration of protein standard solution was added to the 96 hole plate.
3) 10 μL sample was added to the sample hole of the 96 hole plate.
4)300 μL G250 stain was added to each hole.
5) The enzyme was used to detect the absorbance at wavelength 630 nm.
6) Make standard curve according to the absorbance of protein standard solution.
7)Clculate the protein concentration in the sample according to the standard curve and the volume of the sample.