Yeast Spliceosome Analysis Experiment
Yeast Spliceosome Analysis Experiment
A precursor is a macromolecular ribonucleoprotein complex that splices mRNA precursors in the nucleus of eukaryotic cells. It consists of five snRNPs and a large number of non-snRNP proteins, each of which consists of a snRNA and several proteins. This experiment is from "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Yeast Spliceosome Analysis Experiment
Principle
Precursors are macromolecular ribonucleoprotein complexes that splice mRNA precursors in the nucleus of eukaryotic cells, which consist of five snRNPs and a large number of non-snRNP proteins, each of which consists of a snRNA and several proteins.
Materials and Instruments
Tris HCI solution EDTA TE Sodium acetate SDS Homogenization buffer for RNA Lysis buffer for RNA Tris Alkali Tris EDTA Equilibrium Phenol Ethanol Silanization solution TPM8 buffer mRNA HEPES-KOH R buffer Sampling buffer Deionized formamide Northern blotting hybridization solution Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
Liquid Nitrogen Centrifuge Homogenizer Rotary Vacuum Concentrator Test Tubes Centrifuge Tubes Glass Plates PTFE Gaskets Combs Yellow Tape Electrophoresis Tanks Power Supply Flat Tip Capillary Tips Filter Paper
1. Preparation of competitive RNA from mouse tissues
Unless otherwise stated, all solutions were prepared in sterile distilled water.
(1) DEPC virgin water.
(2) Tris HCI solution: 1 mol/L Tris-HCl (pH 7.4), 0.1 mol/L Tris-HCl (pH 7.4). 1 mol/L Tris-HCl is prepared, autoclaved, and stored at room temperature. 0.1 mol/L Tris-HCl is made by diluting 1 mol/L Tris-HCl solution with sterile water.
(3) 0.5 mol/L EDTA (pH 8.0).
(4) 1X TE: 10 mmol/L Tris-HCI ( pH 7.6), 1 mmoI/L EDTA.
(5) 1 mol/L MgCl2 and 0.1 mol/L MgCl2 were prepared, sterilized, and stored at room temperature; 0.1 mol/L MgCl2 was divided into 500~1000 μl each and stored at -20℃.
(6) 3 mol/L sodium acetate (pH 5.4).
(7) 10% (m/V) SDS.
(8) Homogenization buffer for preparation of competitive RNA: 3 mol/L LiCl, 6 mol/L urea, 20 mmol/L Tris-HCl (pH 7.4), 10 mmol/L MgCl2. 500 ml of buffer is available for extraction of RNA from tissues of 16 mice. Prepare in DEPC-treated water, do not autoclave, and use within 24 days of preparation. store at 4°C.
(9) Lysis buffer for preparation of competitive RNA: 10 mmol/L Tris-HCl (pH 7.4), 1 mmol/L EDTA, 1% (m/V) SDS. aseptic and can be stored at room temperature for several days.
(10) 1 mol/L Tris base, sterilized and stored at room temperature.
(11) Tris/EDTA equilibrium phenol, can be stored at 4℃ for 1~2 days. Long-term storage should be at -20℃. Discard the organic phase when it turns pale orange.
(12) Chloroform : Isoamyl alcohol (24 : 1), store at room temperature in a milled flask.
(13) 7.5 mol/L ammonium acetate, prepared and sterilized by filtration through a 0.2 μm pore size filter and stored at 4 °C.
(14) 70% ethanol, stored at room temperature.
2. Solution for spliceosome analysis
(1) Silanization solution: 15% dichlorodimethylsilane dissolved in chloroform, kept in a fume hood in a glass bottle with a ground stopper.
(2) 1 mol/L magnesium acetate, prepared and sterilized by filtration through a 0.2 μm pore size filter and stored at 4°C.
(3) 17 X TP8 buffer, i.e., 850 mmol/L Tris-phosphate, 108 g of Tris base and 20.0 ml of 85% phosphoric acid (analytically pure) were added to 800 ml of sterile water, and then the water was added to 1 L. The pH at 0°C should be 8.0, and the solution should be kept at 4°C after sterilization.
(4) TPM8 buffer: 50 mmol/L Tris-phosphate, pH 8.0 at 0°C, 1.5 mmol/L magnesium acetate.
(5) 20% (m/V) acrylamide (acrylamide: methacrylamide) 50:1: filtered through a disposable 0.2 μm pore size filter and stored for 1 year at 4°C in a sterile, screw-capped bottle.
(6) 10% ammonium persulfate (APS) and TEMED.
(7) Yeast intact cell splice extract.
(8) Radiolabeled precursor mRNA: Radiolabeled actin precursor mRNA can be transcribed in vitro using cloned actin DNA and digested by Hpa II endonuclease as a template. The actin precursor mRNA has 92 nt of exon 1309 nt of intron and 48 nt of exon 2. It is dissolved in water at 40,000~80,000 cpm/μl and stored at -20℃.
(9) Potassium phosphate buffer pH 7.6: To prepare 1 mol/L potassium phosphate storage buffer, dissolve 8.85 g of KH2PO4 and 75.8 g of K2HPO4 in 400 ml of water, adjust the pH of the buffer to 7.6, and then replenish it with water to 500 ml, sterilize it and store it at room temperature. 0.1 mol/L potassium phosphate buffer was made by diluting 1 mol/L potassium phosphate 10 times and divided into 500~1000 cpm/μl. The 0.1 mol/L potassium phosphate buffer was made by diluting 1 mol/L potassium phosphate 10 times, divided into 500~1000 μl each, and stored at -20℃.
(10) 100 mmol/L ATP: 500 mg of ATP was dissolved in 5 ml of water, and the pH was carefully adjusted to 6.8~7.2 with 5 mol/L and then 1 mol/L NaOH. 100 mmol/L ATP was diluted from the master batch with sterile water, and divided into 200~500 μl portions, and stored at -20℃. Before use, melt it quickly in a 37℃ water bath and put it on ice.
(11) 30% (m/V) PEG8000: Weigh 30 g of PEG8000 and add it to 65~70 ml of warm water (50℃), if necessary, heat and stir to dissolve it. After the solution cools, add water to make up to 100 ml and sterilize. Divide into 1 ml or 10 ml portions and store at -20℃.
(12) 1 mol/L HEPES-KOH (pH 7.4): sterilize by filtration through a 0.2 μm pore size filter and store at -20 °C.
(13) R buffer: 50 mmol/L HEPES-KOH (pH 7.4), 2 mmol/L magnesium acetate. 1 ml each at -20℃ or -70℃.
(14) R buffer with competitive RNA: For a 7.5 μl splicing reaction, there should be 7.5 μl of R buffer and 15 μg of competitive RNA (dissolved in 0.5-1 μl). Mix R buffer and competitive RNA before spliceosome analysis.
(15) Dye solution: 10% (m/V) bromophenol blue and xylene blue.
(16) Sampling Buffer: 217 mmol/L Tris-phosphate, pH 8.0 at 0°C, 40% ( V/V) glycerol, 0.25% ( m/V) each of bromophenol blue and xylene blue, plus 2.5 ml of 17X TP8 buffer, 2.5 ml of sterilized glycerol, and 250 μl of dye solution to 4.75 ml of water. Dispense into 1 ml portions and store at -20°C.
(17) 15% ( V/V) methanol, 5% (V/V) acetic acid. Store at room temperature in screw top vials.
3. solution for endogenous snRNP Northern hybridization analysis
(1) 10X TBE: 890 mmol/L Tris-boric acid, 25 mmol/L EDTA ( pH 8.3 ). Store at room temperature.
(2) 1 X TBE, 8 mol/L urea: 89 mmol/L Tris-boric acid ( pH 8.3), 2.5 mmol/L EDTA, 8 mol/L urea.
(3) 0.25 XTBE: made by diluting 10 XTBE with deionized water.
(4) 20X SSCP: 2.4 mol/L NaCl, 0.3 mol/L sodium citrate, 0.40 mol/L sodium phosphate (pH 7.0). Store at room temperature.
(5) Deionized formamide.
(6) 50X Denhardt solution: 1% (m/V) water-soluble polyphenylene enamel ( Ficoll, average molecular mass 400000 Da), 1% ( m/V) polyvinylpyrrolidone (average molecular mass 40000 Da), 1% ( m/V) BSA. filtered through disposable filters with a pore size of 0.45 μm, then divided into 20-50 ml each and stored at -20℃. 20°C. Store at -20°C.
(7) Single-stranded salmon sperm DNA (ssDNA ), 10 mg/ml: dissolve double-stranded salmon sperm DNA 50 μg/μl in 1XTE. sonicate to shear the DNA to break it into 300 bp fragments (analyzed by agarose gel electrophoresis). Boil and cool rapidly, then dilute to 10 μg/μl with TE, dispense and store at -20°C.
(8) Northern blotting hybridization solution: 50% ( m/V) formamide, 0.1% (m/V) SDS, 5X SSCP, 3X Denhardt's solution, 100 μg/ml ssDNA. store at -20°C, and heat up to 60~65°C before use.
(9) 50 mmol/L EDTA (pH 8.0), 0.1 % ( m/V) SDS.
(10) Radiolabeled probe: In this experiment, a double-stranded DNA fragment of the snRNA gene was used to prepare the probe for Northern hybridization. The DNA fragments can be prepared either from cloned DNA digested with endonuclease and purified by agarose gel electrophoresis, or by PCR amplification. snRNA gene DNA fragments range from 200 to 1300 bp in length, and the DNA fragments are radiolabeled by a random 6-polydeoxyoligonucleotide primer reaction, and then amplified with Klenow polymerase and radionucleotide (2.5 μl). [ α-32P ] dATP, for 3000 Ci/mmol, 10 mCi/ml ), 25 μl reaction system. The reaction was terminated by adding 225 μl of 50 mmol/L EDTA, 0.1% SDS. The probe was denatured by heating at 100°C for 10 min and then immediately added to 15 ml of hybridization solution at 65°C.
(11) 3X Northern Blot Wash Buffer: 3X SSCP, 0.1% SDS.
(12) 0.1X Norhern Blot Wash Buffer: 0.1X SSCP, 0.1% SDS.
4. Equipment
(1) Liquid Nitrogen ( N2 ).
(2) Centrifuge, homogenizer, rotary vacuum concentrator.
(3) Various test tubes and centrifuge tubes.
(4) Glass plates: one is 26.5 cm high, 20.2 cm wide and 0.4 cm thick. The glass plate with a notch has the same dimensions as the previous one, but with a notch 2.2 cm deep and 16.3 cm wide.
(5) PTFE spacer and comb: the spacer and comb are 0.5 mm thick, the comb is 16 cm wide and 2.7 cm high, and has 14 teeth, 8 mm wide and 11 mm long, with 3 mm spacing between teeth.
(6) Yellow tape.
(7) Electrophoresis bath.
(8) Power supply: a power supply capable of delivering 200 V and 50 mA, constant voltage.
(9) Miniature pulsed peristaltic pump or equivalent.
(10) Flat tip capillary tip.
(11) Whatman 3 MM filter paper.
(12) Rigid glass or stainless steel dish (for soaking gel and washing Nonhern blot): wash with soap and water, dry bake at 252°C for 4-5 h. Then rinse with deionized water and air dry.
(13) Electrotransfer device: two gels can be placed at the same time and the device is placed in a cold room.
(14) UV crosslinker: three 15 W UV lamps spaced 8.5 cm apart from each other and 35 cm from the sample plane.
(15) Microwave oven or hot plate.
(16) Hybridization bag and sealer.
(17) Water bath shaker or hybridization oven.
(18) X-rays, pressurized cassettes, sensitizing screens, and film processing equipment.
(19) Gel dryer (optional).
II. Methods of Operation
1. Preparation of competitive RNA for spliceosome analysis
(1) Wear dust-free gloves to avoid contamination of RNase on your hands.
(2) Remove small intestine and liver from 8-16 mice. Execute one mouse at a time and remove its small intestine and liver. Avoid removing the gallbladder and pancreas as these organs are rich in RNase. Removed small intestines and livers should be immediately placed in liquid nitrogen, where they can be stored overnight at -70°C for up to 1 year.
(3) Prepare the homogenate. Thaw several pieces of small intestine on a clean paper towel at room temperature. Squeeze out as much residue as possible from the small intestines, cut the small intestines into 5.08-7.62 cm segments, and transfer them to 50 ml sterile disposable polypropylene centrifuge tubes filled with 10 ml ice-cold homogenization buffer. Use 10 ml of homogenization buffer per mouse uterus; if the buffer is too low the yield of RNA will be low. Liver can be added directly to the buffer.
(4) Before homogenization, wash the homogenizing head with DEPC-treated water and ethanol. Homogenize 20-30 ml of sample at a time, and homogenize the liver sample at medium speed for 1 min and the small intestine for 30 s. The homogenization time requirement is not stringent, but depends mainly on the homogenization situation. The homogenate should be very thick, with no visible tissue mass. When finished, the homogenate is left at room temperature.
(5) Centrifugation to remove residue. The homogenate is transferred to a centrifuge tube and centrifuged at 8500 g for 15 min at room temperature to remove the residue.
(6) Precipitate RNA: Transfer the homogenate to a new centrifuge tube, avoiding aspiration of the 1-2 ml of viscous material in the upper layer of the supernatant and the residue at the bottom. Place the homogenate on ice in a cold room overnight (at least 6 h, up to 2-3 days). The RNA precipitate was centrifuged at 11,000 g for 15 min at 4°C in a high-speed centrifuge.
(7) Dissolve RNA and extract with organic solvent. Dissolve the RNA precipitate in 2.5 ml of lysis buffer and transfer to a corex centrifuge tube. Wearing gloves and goggles and a protective suit, add an equal volume of phenol to the dissolved RNA. Vortex the aqueous phase and the phenol solution and centrifuge at 11,000 g for 10 min at 4°C in a high-speed centrifuge to separate the aqueous phase from the yellow phenol layer, and transfer the aqueous phase containing the RNA to a clean Corex tube. The aqueous phase was transferred to a clean Corex tube. 1 ml of fresh lysis buffer was added to the phenol layer, re-extracted, and the aqueous phase was combined. Re-extract the combined aqueous phases with an equal volume of fresh phenol. Add 1 to 2 times the volume of chloroform: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 by adding 1/15 volume of 3 mol/L sodium acetate (pH 5.4) to the aqueous phase, followed by 3 times the volume of anhydrous ethanol, mixing, and allowing to stand on dry ice for 10 min or -20°C overnight. Centrifuge at 11000 g at 4°C on a high-speed centrifuge and discard the supernatant. Wash twice with 70% ethanol at room temperature, aspirate 70% of the ethanol, and then aspirate the remaining ethanol on a clean paper towel.
(9) Dissolve and reprecipitate RNA. Dissolve RNA in cold water, 0.25 ml per organ, on ice. For 16 mice, there should be 3-4 ml of RNA solution per organ. Add 7.5 mol/L ammonium acetate to the RNA solution to a final concentration of 0.2 mol/L, mix, and divide into 400 μl per centrifuge tube. Add 1.2 ml of anhydrous ethanol to each tube and store at -70 °C.
(10) To prepare competitive RNA, centrifuge 2 or 3 tubes of precipitated RNA (14000~15000 g for 10 min) at 4℃. Wash twice with 70% ethanol at room temperature and dry the precipitate with a rotary vacuum concentrator. Dissolve the RNA in a small volume of water and combine the RNA in a total volume of about 100~150 μl. Measure the concentration and purity of the RNA (take out a small amount, dilute it 500 or 1,000 times, and measure the light absorption values at λ260 and λ280, λ260 1OD is equivalent to 40 μg/ml RNA). The mother liquor concentration of competitive RNA should be 20-30 μg/μl. RNA can be used for analytical experiments when OD260/OD280 is higher than or equal to 1.6. This solubilized RNA can be stored at -70℃ for two years.
2. Filling non-denaturing gel
(1) Wash the glass plate, comb, spacer and silanized glass plate with notch, do not silanize another glass plate, otherwise the gel will be damaged when the glass plate is separated after electrophoresis.
(2) Mount the glass plate and spacer together, lay the glass plate without notch flat and place the spacer with one end of the spacer flush with the bottom of the gel, place the glass plate with notch on top with the silanized side facing in, clamp from top to bottom, wrap the bottom of the two plates and both sides with yellow tape and remove the clamps.
(3) Filling the gel. Place the mounted glass plates on absorbent paper at a slight angle to minimize the hydrostatic pressure of the filled gel. Prepare one or two 3.2% gels by adding the following to a 150 ml sterile flask in the following order: 61.5 ml room temperature water, 4.8 ml 17X TP8, 12.8 ml acrylamide storage solution. Stir gently and slowly add 120 μl of 1 mol/L magnesium acetate, 120 μl TEMED, and 660 μl of 10% APS. pour the mixture into the trough 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. Clamp with screwdrivers so that the glass plates and comb are pressed together, and pour some more gel onto the comb. Allow the gel to polymerize for 1~4 h at room temperature.
(4) At least 1 h before starting the splicing reaction, load the gel in an electrophoresis tank with a circulation pump in a cold room. Pour in the electrophoresis buffer so that the bottom of the glass plate is immersed for 2.5 cm and the top of the gel is immersed for at least 1 cm. Attach 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 spiking wells with buffer. Pre-electrophoresis at 160 V for half an inch before sampling, starting the current at 12.5 mA, then dropping to 11 mA and maintaining a constant current.
3. Analysis of basic spliceosomes
(1) Prepare tubes with R buffer and competitive RNA. Add 6 μl of Competitive RNA Stock Solution (20 μg/μl) to 60 μl of R Buffer, mix, and add 8.3 μl of each to six 1.7 ml centrifuge tubes placed on ice.
(2) Add the following solutions to the centrifuge tubes in order to prepare the splicing reaction (on ice): 13.8 μl of H2O, 1 μl of 100 mmol/L DTT, 3 μl of 1 mol/L potassium phosphate buffer, 5.0 μl of 30% PEG8000, 1.5 μl of 0.1 mol/L MgCl2, 1 μl of 100 mmol/L ATP, 20 μl of Intact cell extracts were mixed and centrifuged for 2 s. For samples at time point 0, 6.5 μl of the reaction mixture was removed and added to the first tube containing R buffer and competitive RNA and placed on ice (radiolabeled precursor mRNA was added to this sample later).
(3) Initiation and termination of the splicing reaction. The reaction mixture was placed in a water bath at 23 ℃ for 2~3 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 mmol/L HEPES, and 2 mmol/L ATP. /L HEPES, 80 μmol/L EDTA, 2.2 mmol/L DTT, 8% ( V/V) glycerol, 0.4 nmol/L precursor mRNA, and 40-80 μg of extract. After addition of radiolabeled precursor mRNA, 7.5 μl of reaction solution was removed every 1 min and added sequentially to the centrifuge tubes containing R buffer and competitive RNA, the samples (in R buffer and competitive RNA) were kept on ice, and the last removed samples (in R buffer and competitive RNA) were allowed to incubate for 10 min on ice while 1 μl of radiolabeled actin precursor mRNA was added to the samples of 0 time point. Add 1 μl of radiolabeled actin precursor mRNA to the samples at time 0. After 10 min, add 4 μl of ice-cold Sampling Buffer to each tube of sample, mix quickly, and centrifuge in a cold room for 2 s. Keep samples on ice until they are ready to be sampled.
(4) Sampling and electrophoresis. Turn off the power and circulating pump, rinse the sample wells quickly, but keep the gel strip between the sample wells in an upright position. Pipette 15 μl of each sample with a flat-tip micropipette, extend the tip to the bottom of the wells to add the sample, and avoid touching the sample in the wells. 150~160 V electrophoresis is performed for 14~22 h, and the circulating pump is turned on to circulate the buffer for 30 min to 1 h. The buffer is then pumped to the bottom of the wells to add the sample.
(5) Remove the gel. Before removing the gel, cut two pieces of Whatman 3 MM filter paper of the same size as the notched glass plate and place them next to the gel. Turn off the power and the circulation pump, dismantle the electrophoresis unit at room temperature, remove the tape and gaskets, and carefully remove the notched glass plate; the gel will be left on the non-notched glass plate. Then place a piece of 3 MM filter paper on top of the gel and press gently with gloves so that the gel is firmly attached to the filter paper. Place another piece of filter paper on top, flip the glass plate with the gel on it, remove the glass plate, and hold the gel with the filter paper.
(6) Expose the gel to X-rays. Wrap the gel and filter paper in plastic wrap and expose the x-rays at -70°C (requires intensifier screen). The gel can be thawed/frozen several times to expose several X-rays. After exposure, the gel can be frozen at -20°C for later manipulation (drying or transferring RNA to a nylon membrane).
(7) Multiple exposures or exposures to phosphor plates precipitate the RNA in the gel and allow the gel to dry. Remove the plastic film and the second filter paper, soak the gel bound to one filter paper in sufficient 15% methanol and 5% acetic acid, and shake slowly at room temperature for 20 min. The gel and the filter paper will separate, but as soon as the fixative is removed, they will bind. Remove the fixative slowly with a pipette with a vacuum pump and gently transfer the gel and filter paper to two sheets of 3 MM filter paper wearing gloves. Dry the gel in a gel dryer at 60°C for 45-60 min.
(8) Clean the electrophoresis tank. During electrophoresis, the platinum wire in the buffer tank may precipitate. Wipe and rinse with deionized water to remove the precipitate, and air dry.
4. Nonhern blot analysis of endogenous snRNP complexes
(1) When analyzing RNA-binding proteins by nondenaturing electrophoresis, it is essential to determine by preexperimentation the optimal conditions for efficient separation of the RNA complex from other unbound factors. For example, precursor mRNAs in splicing-dependent complexes can be separated from unbound snRNPs by Northern blotting. In initial experiments, the splicing-dependent complex can be traced by radiolabeled precursor mRNA, while the location of the unbound snRNP can be determined by hybridization to a snRNP-specific probe. In subsequent experiments to determine which snRNAs are present in the splicing-dependent complex, transcripts with little or no radioactivity are used in nondenaturing gel analyses, but the gel treatments used for Northern blotting are the same as those presented here.
(2) Treatment of the gel (to make it suitable for transferring RNA to the membrane). After the gel has been exposed to X-rays, remove the plastic film and the second 3 mm filter paper, transfer the gel bound to the first filter paper to a heat-resistant glass dish containing a sufficient amount of 1X TBE, 8 mol/L urea, with the liquid level about 2 cm above the surface, and shake slowly at room temperature for 15 min. Carefully remove the filter paper and remove the liquid with a pipette attached to the vacuum pump, being careful to avoid damaging the gel, and add a fresh 1X TBE, 8 mol/L urea, 8 mol/L urea, 8 mol/L urea, and a new 1X TBE, 8 mol/L urea, 8 mol/L urea, 8 mol/L urea. Add fresh 1X TBE, 8 mol/L urea, and shake for another 15 min while cutting four 3 MM filter papers of the same size as the gel. After 15 min, place two 3 MM filter papers under the gel, pipette off the liquid, and keep the disk level so that the gel is intact on top of the filter papers. Soaking in 8 mol/L urea is important for efficient transfer of snRNA and precursor mRNA from the complex to the membrane.
(3) Attach the gel and nylon membrane to the transfer device. Let the nylon membrane be wetted in water and then submerged in 0.25X TBE. Load the gel, nylon membrane, and filter paper onto the electro-transfer device ( pay attention to the orientation to transfer RNA from the gel to the membrane).
(4) Transfer at 115~120 V for 1 h. The temperature of the buffer will rise to nearly 37°C after 1 h of transfer.
(5) Cross-link the RNA to the membrane. Remove the electro-transfer device and remove the filter paper and gel from the membrane. Mark the membrane with a ballpoint pen to show the side of the membrane in contact with the gel during transfer. Quickly rinse with fresh 0.25X TBE to remove gel debris and place the nylon membrane on a clean glass dish with the marked side facing up. Wrap the nylon membrane and the glass dish with a layer of plastic wrap. Avoid letting the membrane dry out, as the RNA and membrane cross-link most easily when the membrane is wet. Place the glass dish 35 cm from the UV lamp and turn on the UV lamp for 12 min.
(6) Expose the membrane to X-rays to obtain an image of the radiolabeled precursor mRNA and to check the transfer efficiency. If radiolabeled precursor mRMA is used in the reaction, the transfer efficiency can be checked by autoradiography. Allow the membrane to dry in air for 30-60 min. Hold the nylon membrane with a piece of 3 mm filter paper with the labeled side facing up. Wrap the nylon film and the filter paper in a layer of plastic wrap and expose the X-ray film (in a cassette) at -70°C.
(7) Pretreat the nylon membrane by heating in low salt and SDS. Add 500 to 1000 ml of 0.1X blotting wash solution to a rigid heat-resistant glass dish, cover with plastic film, and bring to a boil in a microwave oven. Carefully submerge the membrane in the solution, cover with plastic film, and boil in a microwave oven (low heat) for 10 min. Shake slowly for 10 min at room temperature.
(8) Pre-hybridization. Hold the nylon membrane with two pieces of 3 MM filter paper of the same size as the nylon membrane and place them together in a hybridization bag, carefully remove the filter paper and leave the membrane in the bag. Add about 20 ml of hybridization solution (no probe) to the hybridization bag. Lay the bag flat on a table, squeeze out all the air bubbles (they will prevent the probe from hybridizing), and seal the bag. Incubate for at least 1 h at 42°C in a shaking water bath (slow shaking).
(9) Preparation of hybridization probes is described in the Experimental Materials section. Remove the hybridization bag from the water bath, cut a corner and pour out the hybridization solution. Pipette the hybridization solution containing the probe, squeeze out the air bubbles (important) and seal the hybridization bag. Incubate the bag in a shaking water bath at 42℃ for 12-24 h.
(10) Wash the membrane. Cut off one corner and pour the hybridization solution into the radioactive waste tank. Place the hybridization bag flat on several pieces of paper towels (with absorbent paper underneath), cut open the bag, and use flat-tipped tweezers to pull out the hybridization membrane and place it into a hard glass dish containing 500 ml of 3X blotting solution. Shake at room temperature for 10 min, pour the wash solution into the radioactive waste solution reservoir and repeat the wash twice. For the fourth wash, add 500 ml of 3X Blotting Wash at 55°C and incubate for 10 min in a 55°C water bath. For the final wash, add 500 ml of 0.1X Blotting Wash at 55°C and incubate for 10 min in a 55°C water bath.
(11) Exposure. Remove the hybridization film from the wash solution, blot it with a 3 mm filter paper, and dry it in air for 30 min~1 h. Hold the hybridization film with a piece of 3 mm filter paper, with the marked side facing upwards, and wrap it with a piece of plastic film. The films were exposed to X-rays at -70℃ for 12 h~5 days in a pressurized dark box.
(12) Remove the probe and hybridize with another probe. The hybridization film can be hybridized with at least 6 probes in sequence. After exposure, the original probe can be removed by the method in step (7). To check the efficiency of probe removal, the film can be allowed to dry in air and then exposed to X-rays before hybridizing with the next probe.