Yeast splice experiment operation method - Database & Sql Blog Articles

1. Prepare competitive RNA for splicing analysis (1) Wear dust-free gloves to avoid contamination of RNase in the hands. (2) The small intestine and liver were taken out from 8 to 16 mice. One mouse was sacrificed at a time and its small intestine and liver were removed. Avoid gallbladder and pancreas during operation because these organs are rich in nucleases. The removed small intestine and liver should be immediately placed in liquid nitrogen, stored at -70 ° C overnight, and stored for up to 1 year in liquid nitrogen. (3) Prepare the homogenate. Thaw a few small intestines on a clean paper towel at room temperature. The residue in the small intestine was squeezed as much as possible, and the small intestine was cut into small pieces of 5.08 to 7.62 cm and transferred to a 50 ml sterile disposable polypropylene centrifuge tube supplemented with 10 ml of ice-cold homogenization buffer. 10 ml of homogenization buffer was used for each mouse's uterus, and if the buffer was too little, the yield of RNA would be very low. The liver can be added directly to the buffer. (4) Wash the homogenate head with DEPC treated water and ethanol before homogenization. The sample can be homogenized in 20~30 ml at a time, the liver sample is homogenized at medium speed for 1 min, and the small intestine is homogenized for 30 s. The homogenization time requirement is not strict, mainly depending on the condition of the homogenate. The homogenate should be thick and there are no visible tissue blocks. At the end, the homogenate was placed at room temperature. (5) Remove the residue by centrifugation. The homogenate was transferred to a centrifuge tube and centrifuged at 8500 g for 15 min at room temperature to remove the residue. (6) Precipitating RNA. Transfer the homogenate to a new centrifuge tube to avoid absorbing 1~2 ml of the supernatant and the bottom residue from the supernatant. The homogenate is placed on ice in the cold room overnight (at least 6 h, up to 2 to 3 days). The RNA was pelleted by centrifugation at 11,000 g for 15 min at 4 ° C using a high speed centrifuge. (7) Dissolve RNA and extract with an organic solvent. Dissolve the RNA pellet in 2.5 ml lysis buffer and transfer to a corex centrifuge tube. Put on gloves and goggles, put on protective clothing, add an equal volume of phenol to the dissolved RNA. The aqueous phase and the phenol solution were vortexed, and the aqueous phase and the yellow phenol layer were separated by centrifugation at 11,000 g for 10 min at 4 ° C in a high speed centrifuge, and the RNA-containing aqueous phase was transferred to a clean Corex tube. Add 1 ml of fresh lysis buffer to the phenol layer, re-extract and combine the aqueous phases. The combined aqueous phase was re-extracted with an equal volume of fresh phenol. Add 1 to 2 volumes of isoamyl alcohol to the extracted aqueous phase, vortex to mix, and centrifuge to separate the aqueous and organic phases. Transfer the upper aqueous phase to a clean Corex tube. (8) Precipitate RNA with sodium acetate and ethanol, add 1/15 volume of 3 mol/L sodium acetate (pH 5.4) to the aqueous phase, then add 3 volumes of absolute ethanol, mix and place on dry ice for 10 min or -20 ° C overnight. Centrifuge at 11000 g at 4 ° C in a high speed centrifuge and discard the supernatant. Wash twice with 70% ethanol at room temperature, aspirate 70% ethanol, and aspirate the remaining ethanol with a clean paper towel. (9) Dissolve and reprecipitate RNA. Dissolve RNA in cold water, 0.25 ml per organ, and place on ice. For 16 mice, each device should have 3 to 4 ml of RNA solution. Add 7.5 mol/L ammonium acetate to the RNA solution to a final concentration of 0.2 mol/L. After mixing, divide into 400 μl of each centrifuge tube. Add 1.2 ml of absolute ethanol to each tube and store at -70 °C. (10) Prepare competitive RNA and centrifuge 2 or 3 tubes of precipitated RNA (14000~15000 g, 10 min) at 4 °C. The mixture was washed twice with 70% ethanol at room temperature and the precipitate was dried using a rotary vacuum concentrator. Dissolve RNA with a small volume of water and combine RNA for a total volume of approximately 100-150 μl. The concentration and purity of the RNA were measured (take a small amount, dilute 500 or 1000 times, and measure the light absorption at λ260 and λ280, λ260 1OD corresponds to 40 μg/ml RNA). The mother liquor concentration of competitive RNA should be 20~30 μg/μl. When OD260/OD280 is higher than or equal to 1.6, RNA can be used for analytical experiments. This solubilized RNA can be stored for two years at -70 °C. 2. Fill the non-denaturing gel (1) Clean the glass plate, comb, gasket and silanized glass plate with the notch, do not silanize the other glass plate, otherwise the gel will be destroyed when the glass plate is separated after electrophoresis. (2) Install the glass plate and the gasket together, lay the glass plate without the notch flat, and place the gasket. One end of the gasket is flush with the bottom of the gel, and the glass plate with the notch is placed. Above, the silanized side is facing inward, clamped from top to bottom, and the bottom and sides of the two plates are wrapped with yellow tape to remove the clip. (3) Fill the gel. Place the mounted glass plate on absorbent paper and tilt it slightly to reduce the static pressure of the gel after filling. Prepare one or two 3.2% gels and add the following to a 150 ml sterile flask: 61.5 ml of room temperature water, 4.8 ml of 17X TP8, 12.8 ml of acrylamide stock solution. Gently stir and slowly add 120 μl of 1 mol/L magnesium acetate, 120 μl TEMED and 660 μl 10% APS. Pour the mixture into the grooves formed by the two glass plates until the liquid level reaches the top of the notched glass plate and insert the comb so that each tooth is fully immersed in the gel. The clamp is clamped so that the glass plate and the comb are pressed together, and then some gel is poured on the comb. Allow the gel to polymerize at room temperature for 1 to 4 hours. (4) At least 1 h before the start of the splicing reaction, the gel is placed in an electrophoresis tank with a circulation pump in a cold room. Pour in the running buffer to immerse the bottom of the glass plate at 2.5 cm and the top of the gel at least 1 cm. Install and connect the two tubes of the peristaltic pump to the bottom and top buffer tanks to circulate the buffer from the bottom tank to the top tank. Gently rinse the well with buffer. The 160 V pre-electrophoresis half-inch before loading, starting at 12.5 mA, then dropping to 11 mA and maintaining a constant current. 3. Analysis of basic splices (1) Prepare tubules with R buffer and competitive RNA. Add 6 μl of competitive RNA stock solution (20 μg/μl) to 60 μl of R buffer. After mixing, add 8.3 μl to each of the six 1.7 ml centrifuge tubes and place the tubes on ice. (2) Add the following solution to the centrifuge tube in order to prepare the splicing reaction (on ice): 13.8 μl H2O, 1 μl 100 mmol/L DTT, 3 μl 1 mol/L potassium phosphate buffer, 5.0 μl 30 % PEG8000, 1.5 μl 0.1 mol/L MgCl2, 1 μl 100 mmol/L ATP, 20 μl intact cell extract, mix and centrifuge for 2 s. For the sample at time 0, 6.5 μl of the reaction mixture was taken and added to the first tube containing R buffer and competitive RNA, placed on ice (radiolabeled precursor mRNA was added to this sample later). (3) Start and terminate the splicing reaction. The reaction mixture was placed in a 23 ° C water bath for 2 to 3 min, and then 5.5 μl of radiolabeled actin precursor mRNA was added. The concentrations of the components in the reaction system were: 60 mmol/L phosphate, 3% (m/V) PEG6000, 3 mmol/L MgCl2, 2 mmol/L ATP, 20 mmol/L KCl, 8 mmol/L HEPES, 80 μmol/L EDTA, 2.2 mmol/L DTT, 8% (v/V) glycerol, 0.4 nmol/L precursor mRNA, 40-80 μg extract. After adding the radiolabeled precursor mRNA, take 7.5 μl of the reaction solution every 1 min and add it to the centrifuge tube containing R buffer and competitive RNA. The sample (R buffer and competitive RNA) is kept on ice. The removed samples (R buffer and competing RNA) were incubated on ice for 10 min while 1 μl of radiolabeled actin precursor mRNA was added to the sample at time 0. After 10 min, 4 μl of ice-cold was added. Load the buffer into each tube sample, mix quickly, and centrifuge for 2 s in the cold room. Samples should always be placed on ice before loading. (4) Loading and electrophoresis. Turn off the power and circulation pump before loading and quickly rinse the sample well, but the gel strip between the wells should be kept upright. Use a flat microtip to draw 15 μl of each sample, and the tip is extended to the bottom of the well. Avoid touching the sample in the well. 150~160 V electrophoresis for 14~22 h. After electrophoresis for 30 min to 1 h, the circulating pump was turned on to make the buffer flow. (5) Remove the gel. Before taking the gel, cut two Whatman 3 MM filter papers the same size as the notched glass plates and set them aside. Turn off the power and circulation pump, remove the electrophoresis device at room temperature, remove the tape and gasket, carefully remove the notched glass plate, and leave the gel on the unrecessed glass plate. Then put a piece of 3 MM filter paper on the gel, gently press the gel to connect the gel tightly to the filter paper, put a piece of filter paper on top, flip the glass plate with gel, remove the glass plate, and use the filter paper tray Live the gel. (6) Expose the gel to the X-ray film. Cover the gel and filter paper with a plastic wrap and expose the X-ray film at -70 °C (intensifying screen required). The gel can be thawed/frozen several times and exposed to several X-ray films. After exposure, the gel can be frozen at -20 ° C for later processing (drying or transferring RNA onto a nylon membrane). (7) Multiple exposures or exposure to the phosphor plate to precipitate the RNA in the gel and dry the gel. Remove the plastic film and the second filter paper, soak the gel combined with a piece of filter paper with a sufficient amount of 15% methanol and 5% acetic acid, and shake slowly for 20 min at room temperature. The gel and filter paper will separate, but once the fixative is removed, the two will combine. Slowly aspirate the fixative with a pipette with a vacuum pump and gently transfer the gel and filter paper to the two 3 MM filter papers with gloves. Dry in a gel drier at 60 ° C for 45-60 min. (8) Clean the electrophoresis tank. During electrophoresis, the platinum wire of the buffer tank will precipitate, wipe and rinse with deionized water to remove the precipitate and air dry. 4. Nonhern blot analysis of endogenous snRNP complexes (1) It is necessary to determine the optimal conditions for efficient separation of RNA complexes from other unbound factors by pre-experimental analysis of RNA-binding proteins by non-denaturing electrophoresis. For example, the precursor mRNA in the splicing-dependent complex can be separated from the unbound snRNP by Northern blotting. In the initial experiments, the splicing-dependent complex was traced by radiolabeled precursor mRNA, and the location of the non-snRNP binding was determined by hybridization with a snRNP-specific probe. In subsequent experiments to determine which snRNAs are present in the splicing-dependent complex, very low or no radioactive transcripts are used in non-denaturing gel analysis, but the gel treatment for Northern blotting is described here. same. (2) Treat the gel (making it suitable for transferring RNA to the membrane). After the gel is exposed to the X-ray film, the plastic film and the second 3 MM filter paper are removed, and the gel combined with the first filter paper is transferred to a heat-resistant glass plate containing a sufficient amount of 1X TBE and 8 mol/L urea. The liquid level is about 2 cm high and the temperature is slowly shaken for 15 min at room temperature. Carefully remove the filter paper and aspirate the liquid with a suction nozzle connected to the vacuum pump. Carefully operate to prevent damage to the gel. Add a new 1X TBE, 8 mol/L urea, shake for another 15 minutes, and cut 4 sheets of the same size as the gel. MM filter paper, after 15 min, hold two 3 MM filter paper under the gel, aspirate the liquid, keep the surface level, and place the gel completely on the filter paper. Soaking in 8 mol/L urea is very important for the efficient transfer of snRNA and precursor mRNA from the complex to the membrane. (3) Install the gel and nylon membrane on the transfer device. Allow the nylon membrane to be wetted in water and then immersed in 0.25X TBE. Place the gel, nylon membrane, filter paper, etc. on the electrotransfer device (note the direction to transfer RNA from the gel to the membrane). (4) Transfer 1 h at 115~120 V. After 1 h of transfer, the temperature of the buffer rose to approximately 37 °C. (5) Crosslink RNA and membrane. Remove the electrotransfer device and remove the filter paper and gel from the membrane. A mark was placed on the film with a ballpoint pen to show the side that was in contact with the gel during transfer. Rinse quickly with fresh 0.25X TBE to wash off the gel fragments and place the nylon membrane on a clean glass dish with the marked side facing up. Wrap the nylon membrane and glass dish with a plastic film. Avoid drying the membrane because RNA and membranes are most easily crosslinked when the membrane is wet. Place the glass dish 35 cm away from the UV lamp and turn on the UV lamp for 12 min. (6) Exposing the film to the X-ray film to obtain an image of the radiolabeled precursor mRNA and checking the transfer efficiency. If a radiolabeled precursor mRMA is used in the reaction, the efficiency of the transfer can be checked by autoradiography. Allow the film to dry in air for 30 to 60 minutes, and hold the nylon membrane with a 3 MM filter paper with the marked side facing up. Wrap the nylon membrane and filter paper with a plastic film and expose it to the X-ray film at -70 °C (in the tablet). (7) Heat in low salt and SDS to pretreat the nylon membrane. Add 500~1000 ml of 0.1X blotting solution to a hard heat-resistant glass dish, cover with a plastic film, and boil in a microwave oven. Carefully immerse the membrane in the solution, cover with a plastic film, and cook in a microwave oven (low heat) for 10 min. Shake gently for 10 min at room temperature. (8) Pre-hybridization. Hold the nylon membrane with two 3 MM filter papers of the same size as the nylon membrane, put them together in one of the hybridization bags, carefully remove the filter paper, and leave the membrane in the bag. Add about 20 ml of hybridization solution (without probe) to the hybridization bag. Place the bag flat on the table and squeeze out all the air bubbles (the bubbles will prevent the probe from hybridizing) and then seal the hybrid bag. Incubate for at least 1 h in a shaking water bath (slow shake) at 42 °C. (9) Preparation of the hybridization probe is shown in the experimental material section. The hybrid bag was taken out of the water bath, cut into a corner, and the hybridization solution was poured out. The hybridization solution containing the probe is aspirated, the bubbles are bubbled out (important), and the hybridization bag is sealed. Incubate for 12 to 24 hours in a shaking water bath at 42 °C. (10) Wash the film. Cut off the corner and pour the hybridization into a radioactive waste tank. The hybrid bag was placed flat on several paper towels (with absorbent paper underneath), the hybrid bag was cut, and the hybrid membrane was clipped with a flat tweezers and placed in a hard glass plate containing 500 ml of 3X blotting solution. Shake for 10 min at room temperature, pour the washing solution into the radioactive waste liquid and repeat the washing twice. For the fourth wash, add 500 ml of 3X blotting solution at 55 ° C and simmer for 10 min in a 55 ° C water bath. The last wash was added with 500 ml of 0.1X blotting solution at 55 ° C and shaken in a 55 ° C water bath for 10 min. (11) Exposure. The hybrid membrane was removed from the wash solution, blotted dry with 3 MM filter paper, and air dried for 30 min to 1 h. The hybrid membrane was held with a 3 MM filter paper with the marked side facing up and wrapped in a plastic film. The X-ray film was exposed to -70 ° C for 12 h to 5 days in a compression cassette. (12) Remove the probe and hybridize with another probe. The hybridization membrane can hybridize to at least 6 probes in sequence. After exposure, the original probe can be removed by the method of step (7). To check the efficiency of probe removal, the membrane can be allowed to dry in air before being hybridized with the next probe and then exposed to the X-ray film.