Yeast precursor mRNA splicing extract assay
The splicing of eukaryotic pre-mRNA is a two-step process, in which an intron is cut from the precursor and two exons are joined together to form the mature mRNA. this experiment is based on the "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Yeast precursor mRNA splicing extract assay
Principle
It is typical for splicing reactions to use nuclear extracts, i.e., S100 extracts complementing partially purified fractions of SR proteins or crude extracts, most often extracts from HeLa cells. Precursor mRNA substrates are usually prepared by in vitro transcription using phage polymerase.
Materials and Instruments
tRNA UTP RNA polymerase Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
YP Tris-HCl ETDA SDS Glycerol HEPES Potassium salt DTT PMSF Potassium phosphate buffer D-Sorbitol SB buffer Isotonic solution Lysis solution AGK buffer TBE TE Spermidine Transcription buffer NTP ATP Dichloroacetic acid Sodium acetate RNA elution buffer Ammonium acetate Ethanol Staining solution Protease K solution Sodium acetate-EDTA solution Tris Alkali Equilibrium Phenol per Ammonium sulfate Silanization solution RNasin
Centrifuge Shaker Dounce Homogenizer Porcelain Mortar and Pestle Dialysis Bags Dewar Flasks Cryoprotective Gloves Vacuum Filter Racks Power Supply Glass Plates PTFE Combs and Gaskets Yellow Tape Polypropylene Culture Tubes Quicksep Column Benchtop Oscillator Rotary Vacuum Concentrator
1. Solution for the preparation of splicing extracts
All solutions were prepared in distilled water unless otherwise stated.
(1) YP: Add 20 g of yeast extract and 40 g of peptone (hacto peptone) to 1 L of deionized or distilled water. Add 50 g of anhydrous D-glucose to 70 ml of water and dissolve with stirring on a heating plate, replenish with water to 100 ml to prepare a 50% (m/V) glucose solution. Autoclave the YP in a stoppered 2800 ml Fcrnbach flask or a 4 L flask and store the 50% glucose in a screw cap bottle at room temperature. Add 40 ml of 50% glucose per liter of YP before use. The glucose is added to the YP after autoclaving to prevent caramelization due to prolonged autoclaving.
(2) 1 mol/L Tris-HCl (pH 7.6, pH 7.8, and pH 8.0): add 30.29 g of Tris base to 125 ml of water, followed by 92 ml of 1 mol/L HCl. Titrate the pH to 7.6, 7.8, or 8.0 with 1 mol/L HCl, and add water to 250 ml. Autoclave and store at room temperature.
(3) 4 mol/L NaCl, autoclaved and stored at room temperature.
(4) 0.5 mol/L ETDA (pH 8.0): add 18.6 g of EDTA (as disodium dihydrate) to 75 ml of water to dissolve the EDTA and adjust the pH to 8.0 with 10 mol/L NaOH. add water to 100 ml. autoclave and store at room temperature.
(5) 10% (m/V) SDS, stored at room temperature.
(6) 50% (m/V) glycerol, autoclaved and stored at room temperature.
(7) 1 mol/L MgCl2, autoclaved and stored at room temperature.
(8) 2 mol/L KCl, autoclaved and stored at room temperature.
(9) 1 mol/L HEPES potassium salt ( HEPES-K+ ), pH 7.8 at 23°C: add 119.2 g of HEPES and 20.1 g of KOH to 400 ml of water, add water to a total volume of 500 ml. Dilute a small portion of the sample to 10 mmol/L and test for pH 7.5 at 23°C, adjusting the pH with 2 mol/L HCl if necessary. The pH should be 7.8 at 4°C. Autoclave or filter sterilize and store at -20°C. The solution may be autoclaved and stored at -20°C. The solution may yellow a little after autoclaving, but this does not affect its use.
(10) 1 mol/L DTT: DTT solids are stored at -20°C. This solution is stored at 4°C and should be used within 24 hours.
(11) 0.5 mol/L PMSF, stored at -20°C.
(12) 1 mol/L Potassium Phosphate Buffer (pH 7.6): slowly add 8.85 g KH2PO4 and 75.75 g K2HPO4 to 400 ml of water, which, when dissolved, should have a pH of 7.6, then bring the total volume to 500 ml and autoclave. Store at room temperature.
(13) zymdyase solution: Prepare a 20 mg/ml (pH 7.6) zymdyase solution with 20 mmol/L potassium phosphate, 5% glucose buffer before use. Enzymase-100T ( 100000 U/g ) solid was stored at 4°C. To prepare extracts from 1 L of cell culture, 10 mg of ELISA is required. The enzyme is not completely soluble, but does not interfere with use.
(14) 2 mol/L D-sorbitol: autoclave and store at room temperature.
(15) SB buffer: 1 mol/L sorbitol, 50 mmoI/L Tris-HCl ( pH 7.8), 10 mmol/L MgCl2, stored at room temperature. Add 1.5 ml of 1 mol/L DTT to 50 ml of SB to prepare fresh SB+30 mmol/L DTT. 0.75 ml of 1 mol/L DTT to 250 ml of SB to prepare fresh SB+3 mmol/L DTT.
(16) Isotonic solution: 1 mol/L sorbitol, dilute 2 mol/L sorbitol to 1 mol/L with water.
(17) Lysis solution: 0.1% (m/V) SDS, 50 mmol/L EDTA.
(18) Buffer A: 10 mmol/L HEPES-K+ ( pH 7.8), 1.5 mmol/L MgCl2, 20 mol/L KCl, 0.5 mmol/L DTT, 0.5 mmol/L PMSF. Prepare fresh and place on ice. Add 1 ml of 1 mol/L HEPES-K+ (pH 7.8), 150 μl of 1 mol/L MgCl2, 1 ml of 2 mol/L KCl, and 50 μl of 1 mol/L DTT to 97 ml of sterilized water, and add 100 μl of 0.5 mol/L PMSF just prior to use. note that the half-life of PMSF in aqueous solution is approximately 30 min. A small amount of precipitation will occur upon addition of PMSF but is not detrimental to use.
(19) Buffer D: 20 mmol/L HEPES (pH 7.8), 0.2 mmol/L EDTA, 50 mmol/L KCl, 20% glycerol (V/V), 1 mmol DTT, 0.5 mmol/L PMSF, to prepare extracts from 1 L of cell culture, prepare 2 L of buffer D one day ahead of time. autoclave and cool in cold room overnight. Overnight. Add 1 ml of 1 mol/L DTT and 1 ml of 0.5 mol/L PMSF before dialysis.
(20) AGK buffer: 10 mmol/L HEPES-K+ (pH 7.8), 1.5 mmol/L MgCl2, 200 mmol/L KCl, 10% (V/V) glycerol, 0.5 mmol/L DTT, and 0.5 mmol/L PMSF. add PMSF just before use.
2. Solutions for in vitro transcription and splicing analysis
(1) 10X TBE: 890 mmol/L Tris-boric acid, 25 mmol/L EDTA (pH 8.3). Store at room temperature.
(2) 1X TBE: Dilute 10XTBE to 1XTBE with distilled or deionized water and store at room temperature.
(3) 1XTE: 10 mmol/L Tris-HCl ( pH 7.6), 1 mmol/L EDTA. store at room temperature.
(4) 30% (m/V) PEG8000, dissolve by heating at 50°C, autoclave, cool the solution and dispense into 1 ml and 10 ml portions and store at -20°C.
(5) 1 mol/L spermidine: 1.27 g spermidine trihydrochloride was dissolved in 3.5 ml of sterilized water, the pH was adjusted to 7.6 with 10 mol/L NaOH, and 5 ml of water was added to the solution. stored at -20℃.
(6) 10X transcription buffer: 0.4 mol/L Tris-HCl (pH 7.8), 60 mmol/L MgCl2, 40 mmol/L spermidine. 500 μl each was stored at -20℃.
(7) 0.1 mol/L DTT: Dilute 1 mol/L DTT to 0.1 mol/L with water and store 500~750 μl at -20℃.
(8) 10X NTP: 5 mmol/L each of ATP, GTP, CTP, and 1 mmol/L of UTP (pH 7.0), stored at -20℃. When used, thaw quickly in a 37℃ water bath and place on ice.
(9) 100 mmol/L ATP: Store at -20℃. For use, thaw quickly in a 37°C water bath and place on ice.
(10) Dichloroacetic acid (TCA): Prepare 100% TCA by adding 500 g of TCA to 227 ml of water. dilute 10 or 20 times in a measuring cylinder to prepare 10% and 5% TCA, respectively.
(11) 3 mol/L sodium acetate (NaAc) (pH 5.4): add 40.82 g of sodium acetate dihydrate to 75 ml of water, add glacial acetic acid to adjust the pH to 5.4, and finally add water to 100 ml. autoclave and store at room temperature.
(12) RNA elution buffer: 20 mmol/L Tris-HCl (pH 7.6), 0.5 mol/L NaCl, 10 mmol/L EDTA, 2% (V/V) phenol (Tris EDTA or NaAc-EDTA equilibrium), stored at 4°C. The buffer should be used for the elution of RNA.
(13) 7.5 moI/L ammonium acetate ( NH4Ac ): decontaminate with a 0.2 μm pore size filter and store at 4°C.
(14) 70% ( V/V ) ethanol: store at room temperature.
(15) Staining solution: 10% bromophenol blue, 10% xylene blue, stored at room temperature. Dyes may not dissolve completely, but do not affect use. Shake vigorously before use.
(16) Deionized formamide, pH greater than or equal to 1-10 ml per portion, store at -20°C. If stored for more than 6 months, it must be inspected. Check pH when stored for more than 6 months. If pH is below 6.5, deionize.
(17) Transcription Product Sampling Buffer: 98% formamide, 50 mmol/L EDTA, 0.1% each of bromophenol blue and xylene blue. Store at -20℃.
(18) Proteinase K solution: 2.5 mg Proteinase K dissolved in 1 ml of a mixture of 4% (m/V) SDS, 50 mmol/L Tris-HCl (pH 7.8), 0.25 moI/L EDTA. 1 ml of each is stored at -20°C. Store at -20°C.
(19) tRNA: Dissolve 100 mg of E.coli tRNA in 10 ml of sterile water, 0.5~1 ml each, and store at -20 °C.
(20) Splicing assay termination buffer: Mix 200 μl of tRNA and 1 ml of proteinase K solution. 0.2~0.5 ml per portion, store at -20°C, melt at 37°C and keep at 23~25°C to prevent SDS precipitation during use.
(21) Sampling buffer for splicing analysis: 0.1X TBE, 8 mol/L urea, 0.1% bromophenol blue and xylene blue. Add 0.22 g of urea (ultrapure) to 200 μl of sterile water and heat at 65 °C to dissolve the urea. Add 200 μl of this urea solution to 94 μl of water, 3 μl of 10X TBE, and 3 μl of staining solution (10% Bromophenol Blue, 10% Xylene Blue), which should be used the same day.
(22) Sodium acetate-EDTA solution: 300 mmol/L NaAc (pH 5.4), 50 mmol/L EDTA, 50 mmol/L NaAc (pH 5.4), 100 mmol/L EDTA. store at room temperature.
(23) 1 mol/L Tris base, autoclaved and stored at room temperature.
(24) Tris-EDTA equilibrium phenol, store at 4°C for 1-2 days, for long term storage freeze at -20°C. Discard when the organic phase turns pale orange.
(25) NaAc-EDTA equilibrium phenol: 65 ° C heating 500 g of molecular biology grade phenol and add 0.5 g of 8-hydroxyquinoline, then add 500 ml of 300 mmol / L NaAc (pH 5.4), 50 mmol / L EDTA solution, mixing with a stirrer quickly for 10 min, stop stirring and let the liquid divided into the aqueous phase and the organic phase, the top layer of the aqueous phase, aspirated and discarded, then add 100 ml of NaAc (pH 5.4), 50 mmol / L EDTA solution, with a stirrer quickly mixed for 10 minutes, stop stirring and let the liquid divided into the aqueous phase and organic phase, aspirated and discarded. Add 100 ml of 300 mmol/L NaAc (pH 5.4) and 50 mmol/L EDTA solution, stir rapidly for 5 min, let the liquid split into two phases, discard most of the upper aqueous phase, and discard the upper aqueous phase, and the remaining aqueous phase in the upper layer of the inorganic phase will be about 2 cm thick. 4°C can be stored for 1~2 days, and the long term storage should be frozen at -20°C. The organic phase should be discarded when it turns light orange. The organic phase should be discarded when it turns a light orange color.
(26) NaAc-EDTA equilibrium phenol: chloroform: isoamyl alcohol: add 20 ml of isoamyl alcohol to 480 ml of trichloromethane, and then pour the solution into 500 ml of phenol equilibrated with NaAc-EDTA, stir rapidly for about 5 min, so that the phases can be separated, and store in the same way as Tris-EDTA equilibrium phenol.
(27) Chloroform: isoamyl alcohol (24 :1 ): Handled in a fume hood and wearing gloves, 240 ml of chloroform was poured into a 250 ml glass cylinder and isoamyl alcohol was added to bring the volume up to 250 ml. stored in a glass bottle with a lid and stored in a fume hood.
(28) 1X TBE, 8 mol/L urea: Add 240.2 g of urea to 250 ml of warm water and 50 ml of 10X TBE. Heat and stir at low temperature until the urea is dissolved, pour the solution into a measuring cylinder, cool and replenish with water to 500 ml and store at 4°C. If any urea precipitates, store at 37°C for a few minutes. If any urea precipitates, heat the solution in a water bath at 37°C until the urea is dissolved.
(29) Preparation of acrylamide [ 29% (m/V) acrylamide, 1% (m/V) methacrylamide] with 1X TBE, 8 mol/L urea: To obtain a 5% (m/V) acrylamide solution, add 12.1 g of acrylamide (electrophoretic grade) and 0.4 g of methacrylamide to 250 ml of 1X TBE, 8 mol/L urea, and stir until acrylamide is completely dissolved. completely dissolved. Prepare a 7.5% (m/V) acrylamide solution by adding 18.12 g of acrylamide (electrophoretic grade) and 0.63 g of methacrylamide to 250 ml of 1X TBE, 8 mol/L urea and stirring until the acrylamide is completely dissolved. The two acrylamide solutions can be stored for up to one year at 4°C in a bottle with a screw cap. If precipitation occurs during storage, heat in a water bath at 37°C until the precipitate is dissolved.
(30) 10% (m/V) ammonium persulfate (APS) and TEMED; APS can be stored at 4°C for 1 week; TEMED is stored at 4°C.
(31) Silanization solution: 15% (V/V) dichlorodimethylsilane dissolved in chloroform. Store in a frosted glass vial in a fume hood.
(32) 2% (m/V) NaHCO3.
(33) 15% (V/V) methanol, 5% (V/V) acetic acid, stored at room temperature in bottles with screw caps.
3 Equipment and materials used for the preparation of extracts
(1) Shaker: Requires the ability to use large flasks with a speed of up to 300 r/min and maintained at 30°C to culture yeast cells.
(2) Centrifuges: miniature high-speed benchtop (or clinical) centrifuges and ultracentrifuges with appropriate rotors.
(3) Dounce homogenizers of glass or metal.
(4) 12.7 cm porcelain mortar and pestle: for initial use, treat mortar and pestle with chromic acid solution for 15-30 min, then rinse thoroughly with water. Fill the mortar and pestle with AGK buffer, place the pestle and mortar in the mortar and leave it at room temperature for 30 min. Finally, rinse the mortar and pestle thoroughly with sterilized distilled water and air dry. For use, rinse with deionized water, then rinse with sterilized distilled water and air dry.
(5) Dialysis bag (1.27 cm wide, retained molecular mass >3500 Da) and 4 cm dialysis bag holder. The day before the experiment, the dialysis bags were processed as follows: cut into 30 cm strips, boiled in 4 L of 2% sodium bicarbonate for 15 min, and washed three times with plenty of sterilized water. The treated bags can be stored in a tightly closed container in 70% ethanol at 4°C for several years. About 15-30 minutes before using the bag, remove it from the ethanol and squeeze out the excess liquid, soak it in a few hundred milliliters of sterilized water, and then wash it with more sterilized water. Rinse the inside of the dialysis bag twice with 10~12 ml of fresh sterilized water and immerse it in cold Buffer D.
(6) Two 350 ml Dewar's flasks.
(7) 0.5~10 L liquid nitrogen ( N2 ).
(8) Anti-cryogenic gloves.
(9) An ultra-low temperature (-70~-80°C) refrigerator or liquid nitrogen tank.
4. Equipment and materials for in vitro transcription and splicing analysis
(1) [ α-32P ] uridine triphosphate (UTP): 3000 Ci/mmol and 10 mCi/ml.
(2) T7 or SP6 RNA polymerase.
(3) RNasin: 40 U/μl.
(4) Steel vacuum membrane holder supporting 24 mm filters.
(5) Whatman glass fiber (GF/C) 24 mm filter paper.
(6) Power supply: Maintains a stable voltage of up to 1000 V and 100 mA DC.
(7) Two glass plates (4 mm thick): one measuring 19 cm wide by 16 cm high and the other of the same dimensions but with a 2.2 cm deep by 16.3 cm wide slot.
(8) PTFE combs and spacers. For purification of transcripts, the spacer and comb should be 1.5 mm thick, the comb 16 cm wide, 3 cm high, 7 teeth with 2 mm spacing between teeth, each tooth 1.4 cm high, 1.8 cm wide, each tooth forming a pore with a maximum volume of 80 μl. The comb and spacer for splicing analysis are 0.38 mm thick, the comb 15.5 cm wide, 2.6 cm deep, 14 teeth with 4 mm spacing between teeth, each tooth 8 mm high, 1.8 cm wide. The comb and spacer used for splicing analysis were 0.38 mm thick, 15.5 cm wide, 2.6 cm deep, 14 teeth with 4 mm spacing between teeth, each tooth was 8 mm high and 7 mm wide, and each tooth formed a pore that could hold 15 μl.
(9) Yellow tape.
(10) Disposable sterile polypropylene culture tubes with caps (12 mm X 75 mm or 17 mm X 100 mm) and sterile graduated conical polypropylene centrifuge tubes (50 ml) with screw caps.
(11) Empty Quicksep columns (Isolab, Inc, Akron, OH, USA).
(12) X-ray film (20.32 cm X 25.4 cm), light-proof X-ray film cassette, sensitizing screen, X-ray film processing equipment.
(13) Silanized and baked glass rods, Corex test tubes.
(14) Benchtop oscillator.
(15) Spced-Vac concentrator or lyophilizer.
(16) 3 MM Whatman filter paper (46 cm X 67 cm).
(17) Gel dryer (optional).
II. Methods of Operation
1. Homogenizing protoplasmic spheres to obtain whole cell extracts
To obtain the extract with the maximum possible activity, operate as fast as possible until dialysis.
(1) 1 L of yeast cell culture is incubated overnight until the middle to late logarithmic growth phase. First culture 10-50 ml of yeast in 125 ml or 250 ml flasks overnight and transfer to 1 L YPD the next day. The cultures were incubated overnight at 30°C at 250-300 r/min to reach a density of 3X107-5X107/ml. For yeast strain EJ101, the culture was incubated in YPD at 30°C with a doubling time of 90 min.
(2) Collect yeast cells. Collect the cells by centrifugation at 1200~1500 g for 5 min in a high-speed centrifuge. The cells were resuspended with 100 ml of SB+3 mmol/L DTT and centrifuged again, and then the precipitate was resuspended with 30 ml of SB+30 mmol/L DTT and left at room temperature for 15 min. Incubation in 30 mmol/L DTT is very important for protoplasmic sphere formation. The cells were resuspended with 30 ml of SB+3 mmol/L DTT and transferred to sterile 120 ml Erlenmeyer flasks.
(3) Enzymatic incubation of cells and monitoring of protoplasmic sphere formation. Two portions of 20 μl of cells were taken, one portion was added to 1 ml of isotonic solution, and the other portion was added to 1 ml of lysate, and cell lysis was monitored by measuring the light absorption value of λ500. Add 250 μl of zymolysin-100T solution to the cell culture flask and shake slowly at 30℃ 50~60 r/min for 40 min. remove 20 μl every 10~15 min and add 20 μl to 1 ml of isotonic solution and lysate respectively, and then measure the absorption value of λ500 to monitor the formation of protoplasmic spheres. When the light absorption value in the lysis solution is 20% or less of that in the isotonic solution, it indicates that the enzymatic digestion has met the requirements.
(4) All previous steps are performed in a cold chamber to keep the sample cool. Equipment (including pipettes, rotors, and solutions) should be pre-cooled in the cold room overnight.
(5) Homogenize lysed protoplasmic spheres. Using a sterile silanized glass rod, gently resuspend the protoplasmic spheres in cold Buffer A. For 1 L of culture ( 4X107-5X107 cells/ml), use 8 ml of Buffer A. The protoplasmic spheres are then transferred to a cold 7 ml or 14 ml Dounce homogenizer, which is placed on ice and homogenized 5 to 10 times.
(6) The homogenate was treated with 0.2 mol/L potassium chloride to release splicing factors from the nuclei. Pour the homogenate into a 15 ml graduated sterile disposable conical centrifuge tube and note the volume of the homogenate. If there are a large number of air bubbles in the homogenate, centrifuge briefly (2 min) in a cooled benchtop centrifuge to eliminate air bubbles. The homogenate was poured into a 25 ml glass beaker with a small stirrer placed on ice and stirred slowly (~60-120 r/min) for 10 min. 0.9 times the volume of cold 2 mol/L KCl was added slowly (2 min) with a pipette tip to a final concentration of 0.2 mol/L, and the mixture was stirred slowly for another 30 min.
(7) Precipitate cellular debris and organelles by centrifugation twice, transfer the homogenate to a 10 ml or 30 ml screw-cap Oak Ridge test tube and centrifuge at 33,000 g for 30 min at 4°C to precipitate large debris. The tubes were removed from the rotor in the cold room, and the supernatant was transferred to a thick-walled polycarbonate tube with a screw cap and centrifuged at 100,000 g for 1 h at 4°C. The tubes were then centrifuged at 100,000 g for 1 h at 4°C.
(8) Dialyze the extract. Transfer the centrifuge rotor to a cold chamber, carefully remove the tubes and place them in a tube rack with the cap open. The sample is divided into three layers: the thin top layer includes lipids and lipoproteins; the thick middle layer is a clear pale yellowish-green color and includes most of the splicing actives and some ribosomes; and the bottom layer is a clear pale brown color and includes organelles, ribosomes, and large cellular debris. Remove the dialysis bag from Buffer D. Wearing gloves, squeeze out the excess liquid and close one end with a clamp. Aspirate the yellowish-green intermediate layer of liquid with a cold, sterile Pasteur pipette, handling it carefully to avoid aspirating to the upper or bottom layer. The extract is aspirated into a dialysis bag of approximately 6 to 8 ml. Squeeze the air from the bag and hold the bag with a clamp close to the extract. Place the dialysis bag in a sterile beaker containing 1 L of Buffer D and a sterile magnetic stirrer. The beaker is placed in an ice bath surrounded by ice and the ice bath is placed on a magnetic stirrer in a cold room. buffer D is replaced after 1.5 h and the dialysis is stirred for a total of 3 h. Buffer D may be increased as more extract is added.
(9) Centrifugation removes the precipitate produced during dialysis. After dialysis, the amount of extract may decrease by 1-2 ml. Remove the bag from the dialysis buffer, wipe off the liquid outside of the bag with a piece of gauze, open the top clamp, and aspirate the extract with a cold sterile Pasteur pipette and transfer it to a cold Oak Ridge test tube or several cold microcentrifuge tubes. Centrifuge at 12000-14000 g for 10 min at 4℃.
(10) Preservation of extracts. Dispense 100 μl, 200 μl or 500 μl of extract per tube into microcentrifuge tubes or cryotubes on ice. Quickly freeze and store in liquid nitrogen or -70°C refrigerator.
(11) 1 L of yeast culture yields 6 ml of extract, which contains about 20-30 mg of protein per ml.
2. "Freeze-fracture" method for obtaining whole cell extracts.
(1) Cultivate yeast cells as described above. Collect the cells by centrifugation at 1500 g for 10 min at 4℃.
(2) Prepare the cells. Resuspend the cells in 50-100 ml of cold AGK + PMSF buffer and centrifuge at 1500 g for 10 min at 4°C. Keep the sample cold from this step. Pour out the supernatant and resuspend the cells in 20 ml of freshly prepared cold AGK + PMSF buffer and transfer the cell suspension to a sterile 50 ml polypropylene graduated centrifuge tube. Centrifuge the cells at 1500 g for 10 min at 4°C in a bench-top or high-speed centrifuge. Pour off the supernatant, which consists of approximately 0.5 ml of cellular precipitate, and add 0.4 times the volume of AGK+PMSF buffer, resuspend the cells with a silanized and baked glass rod, and place the suspension, which is very viscous, on ice.
(3) To rapidly freeze the suspension, drop the suspension into liquid nitrogen using a Pasteur pipette to form spheres of 0.2-0.5 cm in diameter. Before freezing the cell suspension, place the mortar and pestle in an empty styrene-foamed ice bath, pour liquid nitrogen into and out of the mortar, and after it stops bubbling vigorously, immerse the mortar half-filled with liquid nitrogen into the liquid nitrogen to a depth of 2 cm. To facilitate the aspiration of viscous cell suspensions, the tip of the Pasteur pipette can be broken off to make the mouth of the pipette larger. With the tip of the pipette at least 5 cm above the surface of the liquid nitrogen (to prevent the suspension from freezing in the pipette), add the suspension dropwise to the liquid nitrogen in the mortar.
(4) After all of the suspension has been frozen into small pellets, grind the extract. Holding the mortar in one hand with insulated gloves, gently grind the pellets frozen in liquid nitrogen to turn them into small fragments, and then grind them to turn them into a powder. When the liquid nitrogen in the mortar evaporates, the suspension can be ground into a very fine powder, the finer the powder the better the activity of the extract. The grinding time is usually 20-30 min, even if the liquid nitrogen evaporates from the mortar during the final stages of grinding. The mantle is immersed in liquid nitrogen to a depth of 1-2 cm by constantly replenishing the ice bath.
(5) Transfer the ground cell powder to a test tube for melting. Cool a small spoon in liquid nitrogen and use it to transfer the ground cell powder to an Oak Ridge test tube with a screw cap at room temperature, allowing the ground suspension to melt on ice, intermittently vortexing the tube.
(6) Continue processing as described in steps (7)-(11) of Operation 1, "Obtaining Whole-Cell Extracts from Plasma Protoplasmic Spheres".
(7) 1 L of cell culture yields about 3~4 ml of extract.
3. Splicing analysis of extracts
(1) Preparation of radiolabeled precursor mRNA for splicing assays
① Transcription
A. The transcription product obtained from the 40 μl in vitro transcription reaction system can be used for 6~10 splicing analyses (10~15 reactions), and the transcription product should be used within two weeks.
B. Prepare DNA template for transcription. Choose the appropriate endonuclease to digest 50 μl of DNA (about 50~100 μg) in 200 μl reaction to prepare linear DNA. add 10 μl of 4 mol/L NaCl to adjust the concentration of NaCl in DNA solution to 0.2 mol/L. Extract the DNA with Tris-EDTA equilibrated with phenol, chloroform: isoamyl alcohol, and then precipitate DNA by adding 3 times the volume of anhydrous ethanol. The DNA was precipitated by adding 3 times volume of anhydrous ethanol. The DNA precipitation was washed with 70% ethanol, dried under vacuum and dissolved in 50 μl of 1X TE. Store at -20°C.
C. Perform transcription reaction. For a 40 μl reaction system, add the following reagents to a microcentrifuge tube at room temperature in the following order: 6 μl of sterile water, 4 μl of 0.1 mol/L DTT, 4 μl of 10X transcription buffer, 4 μl of 10X NTP, 4 μl of 30% PEG8000, 2 μl of RNsin, 4 μl of DNA (1-2 μg), 8 μl of [ α-32P ] UTP and 4 μl of T7 RNA polymerization. UTP and 4 μl T7 RNA polymerase. incubate at 37℃ for 2 h. Add 2 μl RNA polymerase and incubate for 1 h. Add 2 μl RNA polymerase.
D. Determination of radioisotope incorporation efficiency by trichloroacetic acid precipitation. Remove 2 μl of the reaction solution and add 10 μl of E.colo tRNA (10 mg/ml), add 500 μl of 10% TCA and incubate on ice for 10 min. Then add the mixture in a small tube to a glass fiber membrane of a vacuum filtration device, rinse the tube with about 1 ml of 5% TCA and add it to the filter, and wash the membrane twice more with 5 ml of 5% TCA and 5 ml of 95% ethanol. The membrane was washed twice more with 5 ml of 5% TCA and 5 ml of 95% ethanol. After drying, the membrane was placed in a scintillation vial containing 15 ml of water and the intensity of radioactivity was measured using a liquid scintillation counter. A good transcription reaction should have about 2X106 Cerenkov/min per 2 μl. The remaining product can be stored at -20°C for up to 2 days.
E. Prepare samples for electrophoresis. Add an equal volume (40 μl) of Sampling Buffer to the reaction, heat at 65°C for 10 min to denature the RNA and place immediately on ice.
② Denaturing polyacrylamide gel electrophoresis analysis
A. Clean and silanize glass plates with notches, using 1.5 mm thick spacers.
B. Fill the gel. Wearing gloves, take 70 ml of 5% acrylamide solution into a beaker, add TEMED and 0.45 ml of 10% APS and mix well, pour it into the gap between the glass plates of the electrophoresis setup until it is flush with the top of the notched glass plate, avoiding air bubbles during the operation. Insert a comb and leave the gel on the bench to agglutinate for at least 1 h. If not used on the same day, the gel can be stored in a ziplock bag with a wet paper towel at room temperature for 1~2 days.
C. Perform gel electrophoresis. Fill the electrophoresis tank with 1X TBE, keep the voltage constant, and pre-electrophoresis for 20 min at a current of about 60-65 mA, so that the temperature of the gel glass plate reaches 40-45°C (quite hot to the touch), do not allow it to get warmer or the glass plate will break.
D. Sample and electrophoresis. Turn off the power, rinse the sample wells, and then apply the sample. Electrophoresis at 55~60 mA for about 1.5 h until bromophenol blue swims out of the gel. Carefully remove the radioactive buffer from the lower container and dispose of it according to radiation safety procedures. In the upper container there is almost no radioactivity and the buffer is poured into the sink. Remove the spacer and cool the gel-glass plate to room temperature, separate the two glass plates, the gel should be attached to the un-notched glass plate, and wrap the gel and the glass plate in plastic wrap. The gel-glass plate can be stored frozen at -20°C and thawed after 24~48 h to complete the transcription product.
③ Extraction and purification of transcription products
A. Radiation autoradiography. In a darkroom, take an X-ray film on clean paper and place a gel-glass plate (gel side down) on the X-ray film for 2 min. Place a dark box over the gel without covering the upwellings, and flash a small flash for 1-2 s at a distance of 4-5 ft (1 ft = 30.48 cm) away, which will expose the outline of the upwellings on the X-ray film, and which can be used to visualize the transcription product by aligning the gel with the image on the X-ray film. This can be done by aligning the gel and the image on the X-ray film to localize the transcription product. Rinse the X-ray film after 2 min of exposure.
B. Locate and cut off the gel band with the transcription product. Align the X-ray film with the gel-glass plate (gel side up) to localize the transcription product. Wearing gloves, use a clean razor blade to cut off the destination band, avoiding cutting off Dosher's gel. Remove the wrapped plastic film from the band and place the cut out band into a disposable polypropylene tube.
C. Extraction of transcription products by crush-soak method. Squeeze the cut gel into pieces with a sterile silanized glass rod and add 1.5 ml of elution buffer. Seal and shake at 37°C for 4 h (or 4°C overnight). Centrifuge the gel at maximum speed for 10 min at room temperature in a bench-top centrifuge to precipitate the gel fragments, aspirate the supernatant (including radioactive transcription products) and filter through a Quicksep column into a 15 ml Gorex tube. Add 1 ml of freshly prepared extraction buffer to the crushed gel, mix for 15-30 min, and repeat the centrifugation and filtration proc