1. Steps of goal DNA PCR:

1） Building a PCR system.

(Repeat step 1 to build a GFP and UAS DNA PCR system.)

Adding different DNA to different tubes to separate and then send to the PCR machine, which processes through the following steps

2) Perform PCR program.

2. Electrophoresis testing and gel recovery

1. Configure electrophoresis gel.

2. Drop the DNA of SPA1, GFP, and UAS DNA into the opening left by the comb.

3. Energize electrophoresis.

4. DNA extraction from the gel.

1) Configure electrophoresis gel.

In this process, use 0.5g of agarose and 50ml of pure water to complete the mixture. The gel can be extracted by putting the configuration into the laboratory microwave oven and heating it until boiling, and the process of configuring the electrophoresis gel is finished.

2) Drop the DNA of SPA1, GFP,and UAS DNA into the opening left by the comb.

Use the pipette gun to move this DNA from the test tube initially configured to the opening where the electronic comb had been produced by leaving a mark.

3) Energize electrophoresis.

Place the electrophoresis into the experimental flumes with the charger to energize it. After energizing the electrophoresis, the DNA will leave some signs, which can be compared to the markers to determine whether it is the goal DNA.

4) DNA extraction from the gel.

a. After DNA electrophoresis, the gel containing the target DNA fragment is quickly cut under the ultraviolet lamp, and the weight of the gel is weighed (the importance of the empty tube is removed). 100 mg gel is equivalent to 100 L volume, regarded as a gel volume.

b. Add equal-volume Buffer GDP. Bathe in 50 to 55C water for 7-10 min to ensure the gel blocks are entirely dissolved.

c. Centrifuge briefly to collect droplets from the wall of the tube. The Fast Pure DNA Mini Columns-G adsorption column was placed in the Collection Tubes2ml collection tube, and the 750ul sol solution was transferred to the adsorption column and centrifuged at 12,000rpm for 30-60s.

d. Discard the filtrate and place the adsorption column in the collection tube. Add 700 pl Buffer GW (containing ethanol) to the adsorption column. Centrifuge at 12,000 rpm for 30-60 s.

e. Repeat Step 5

f. Discard the filtrate and place the adsorption column back into the collection tube—centrifuge at 12,000rpm for 2 min.

g. Place the adsorption column in a 1.5ml sterilized centrifuge tube, add 20-30 dd water drops to the center of the adsorption column, and place for 2 minute-centrifuge at 12,000rpm for 1min. Discard the adsorption column and store DNA at -20℃.

3. Preparation of culture medium

Add the following things into a measuring cylinder; the total of 500ml cultural medium consists of 200ml liquid culture medium and 300ml solid culture medium (each tangible culture medium should add 2g agar) and then divide every 100ml into a flask and sterilize it in a high-pressure steam cooker. During cooling, fix the hands of the operating students with alcohol and light the Alcohol burner. Sterilize the mouth of the flask with an external flame, and finally, pack the culture medium in a Petri dish in a sterile environment near alcohol, etc., and mark it. After final cooling, seal and invert the solid culture medium.

4. digestion

1. Double digestion AD

2. Mix the reagents

3. Use PCR to reaction

a. Mix the following material, which ends up with a total volume of 50ul.

b. Use a centrifuge to mix reagents.

c. Those reagents are placed in a PCR reaction at 37 degrees for 20 minutes.

d. the AD double enzyme digestion system (BD double enzyme digestion system) and marker were injected successively with a pipette gun in the pre-prepared gel gap. After the injection is complete, cover the electrophoretic apparatus for 20 minutes.

e. After the electrophoresis is complete, the middle part of the gel is lifted out, and a particular instrument is placed to take pictures and observe the luminescence.

f. Cut off the luminous part with a blade and melt it in a test tube.

5. homologous recombination

Steps:

1. Calculate the volume of vector and DNA segment supposed to be used in this experiment.

2. Mix the reagents.

3. Use a PCR machine to help the reaction go on

A. The accurate mass of UAS, GFP is calculated by the equation [0.02*base pair number of the vector]ng. The vector BD's mass is calculated using the equation [0.02*base pair number of the segment]. The vector AD's mass is calculated using the equation [0.04*base pair number of the segment]. Since both outcomes are lower than 1 uL, it is rounded to 1 uL. For the AD group, SPA1 is calculated by the equation [0.02*base pair number of the vector]ng.

B. The above reagents are thoroughly mixed by centrifuge.

C. Those reagents are placed in PCR for 50 Celsius, 30 minutes, and preserved at 4 Celsius degrees.

6. Thermal shock conversion

Steps:

1. Rocking inoculation

2. Coated plate

1. Rocking inoculation

1) Pick two test tubes, open the lid, and add 5 ml of LB reagent to each test tube (add 1000 microliters five times).

2) Add five microliters of BD antibody to each of the two test tubes.

3) Dip the pipette tip into the Escherichia coli bacteria, then directly insert the pipette tip into two test tubes.

4) Put the lid on the test tube and seal the two test tubes.

2. Coated plate

1)Use a pipette to remove 450 μ L of the supernatant from the solution containing the BD (or AD) plasmid system, and then extract a portion of the LB solution to flush the plasmid precipitated below the collection tube.

2) Subsequently, the solution was repeatedly sucked up and down with a pipette, resulting in a homogeneous mixture of the plasmid and solution.

3) Then, use a pipette to transfer the solution above the solid culture medium, and use a sterile coating rod to continuously apply the solution in circles until resistance from the culture medium is felt, proving that the culture medium has fully absorbed the solution.

4) Incubate upside down in a 37C incubator for 12-16h.

7. plasmids isolation

Steps:

1. Mix the bacteria solution.

2. Thallus pyrolysis and precipitate.

3. Collect DNA.

4. Plasmid BD-CRY2-GFP and AD-CIB1.

a. Use a centrifuge to mix BD-CRY2-GFP solution (10000 rpm centrifuge 1 minute) and make a precipitate.

b. Add 250ul buffer P1 into precipitation, use a pipette for intensive mixing, and add 250ul buffer P2 in the solution prepared in the previous step, slowly upside down to crack the BD-CRY2-GFP. Adding 350ul buffer P3, repeat the above step to complete dissolution buffer P2, make white precipitate, 12000 rpm centrifuge 10 minutes.

c. Use a pipettor to transfer the precipitate solution into the adsorption column (do not touch the sediment), 12000 rpm centrifuge for 1 minute, liquid waste removal. Adding 500ul buffer PW1 into the adsorption column, repeat the above step. Adding 600ul buffer PW2, repeat the above step. Repeat the step: 12000 rpm centrifuge 1-minute dry adsorption column, removal of residue. After standing for 10 minutes, add 15ul dd H2O into the adsorption column, let stand at room temperature for 10 minutes, 12000 rpm centrifuge, and use DNA extraction. Discard the adsorption column; DNA product save at -20 degrees centigrade.

d. The plasmid insolation steps of AD-CIB1 are the same as above.

Yeast hybrid

1. Use a PCR apparatus to heat the carrier DNA at 95 Celsius for 5 minutes. Froze the DNA carrier immediately by placing the tube in ice for 2 minutes. Then, repeat this step.

2. Transfer the carrier DNA and another reagent to the clean bench, ensuring the experiment undergoes a sterilized condition. Add 100 uL AH109 competence cell. Then, add 5uL of AD-SPA1 and 5uL of BD-CRY2-GFP to the bottom of the tube. Add 10 uL preconditioned carrier DNA.

3. Use a 30 Celsius metal bath, treat the mixture for 30 minutes, and flip the tube to mix the reagent 15 minutes after it begins. After 30 minutes, use a water bath, treat the mixture with 42 Celsius for 15 minutes, and flip the tube to mix the reagent 7.5 minutes after it begins.

4. Coated plate

a. Centrifuge the double hybrid yeast strain 5000rmp for 40s using a centrifuge, then discard the supernatant on a super purification platform, resuspend 400mL of ddH20, and centrifuge for 30s to discard the supernatant.

b. Bring the yeast into the ultra-clean table again, add 50mL of dd H2O resuspend, blow, and mix well.

c. Then, use a pipette to transfer the solution above the solid culture medium, and use a sterile coating rod to continuously apply the solution in circles until resistance from the culture medium is felt, proving that the culture medium has fully absorbed the solution.

5. Rocking inoculation

Place the culture substrate coated with yeast on a shaker and incubate at 28 ℃ for 48 to 96 hours.

6. Select the transformed single colony and culture it in -2 liquid medium at 28℃ and 200rpm for 48h

7. Take 5μl of the successfully cultured bacterial solution, drop it on -Trp-Leu-His-Ade defect medium and -Trp-leu defect medium, respectively, and culture it at 28℃ under dark and blue light irradiation for 48-96h

8. Using the -Trp-leu-his-Ade defect medium to screen the sensitivity of the two systems with different gradient dilutions under blue light irradiation. Then, the plates of -Trp-leu were simultaneously cultured in the dark, and finally, the two systems of varying gradient dilution were cultured in the night, and the point plates were diluted by a 10-fold gradient (1, 10^-1, 10^-2, 10^-4, 10^-6, 10^-8, 10^-10, 10^-12).

GFP Fluorescence Detection

1. Focusing specimens under bright field microscopy conditions

(1) Turn on the power switch (transmission lighting LED on)

(2) Open the transmission light path baffle (Ts2-FL only)

(3) Set the binocular optical path to 100% (only when the image head interface is installed)

(4) Fully open aperture shutter

(5) Set all ring plates to bright field-of-view positions

(6) Placing a 4x objective in the optical path

(7) Adjust brightness (rotate the projection lighting brightness adjustment knob to adjust the field of view brightness)

(8) Place the specimen and place the part to be observed in the optical path

(9) Specimen focusing

(10) Diopter adjustment

(11) Adjusting pupillary distance

2. Close the transmission light path baffle

Place the transmission light way confused in the shaded position.

3. Place the fluorescent filter block into the optical path

Rotate the fluorescent block turntable and place the required fluorescent filter block into the optical path.

4. Turn on fluorescent lighting

Press the fluorescent lighting switch to turn on the light path's LED unit corresponding to the fluorescent filter block.

5. Observation of specimens

(1) Rotate the fluorescent lighting brightness adjustment knob to adjust the field of view brightness

(2) Place the portion of the specimen to be observed into the optical path

(3) If the specimen needs to be observed and the focus is inaccurate, rotate the fine adjustment knob to focus the sample.

(4) When replacing and observing specimens, check the focus and brightness and adjust them as needed.

6. Turn off the power switch

Set the power switch to the "O" position and turn off the microscope switch.

Assay of β-galactosidase activity

Kit components

Sample treatment

1. Bacteria or cultured cells: first 1200rmp,2min, discard the supernatant; Add 100μl extract, ultrasonic break bacteria (ice bath, power 200W, ultrasonic 3s, interval 10s, repeat 30 times);12000g centrifuge at 4℃ for 10min, take the supernatant and put it on the ice to be measured.

2. Organization: According to the tissue mass (g), extract liquid volume (mL) is 1:5 ~10 ratio, ice bath homogenization. 12000rmp centrifuge at 4℃ for 10min, take the supernatant, and put it on the ice to be measured.

Operation steps

1. the spectrophotometer preheats 30 minutes or more, adjusts the wavelength to 400nm, and zero the distilled water.

2. the treatment of standard liquid: distilled water gradient dilution to 200, 100, 50, 25, 12.5, 6.25, 0 nmol/ml.

3. calculate the gradient dilution concentration.

a. 5u mol/ml = 5000n mol/ml

b. x divided by 5000*4

4. Add each reagent in turn according to the operation table

Activity calculation

1. Establishment of the standard curve.

According to the absorbance of the standard tube (x, minus the OD value of the classic tube with A concentration of 0) and the concentration (y, nmol/ml), the standard curve was established, and △A was put into the standard curve to calculate the amount of product generated by the sample (nmol/ml).

2. Calculation of β-GAL activity.

Calculated by bacterial or cell density:

Unit definition: 1nmol p-nitrophenol production per 10,000 bacteria or cells per hour is one unit of enzyme activity.

β - GAL activity (U / 10⁴ cell) = (y * V reverse total) present sample (500 * V present V sample total) present T = 0.04 * y

Cpr: Sample protein concentration, mg/mL; V inverse total: total volume of the reaction system, 0.5mL; V sample: the sample volume added to the reaction system, 0.05mL; Total V sample: add extraction liquid volume, 1mL; W: sample mass, g; 500: total number of cells or bacteria, 5 million; T: reaction time, 0.5h.