A DNA sequence corresponding to mRNA that can be recovered, cloned, sequenced, and can be used as a probe for hybridization or screening libraries. Source: Compact Laboratory Guide to Molecular Biology (5th Edition)
Operation method
PCR differences in mRNA showed
Materials and Instruments
Human total cellular RNA or poly(A)+ RNA Move For materials, reagents and consumables required for the experiment, please refer to "Others". 1. Digest the DNA in total cellular RNA or poly(A) + RNA: 50 ug RNA 10 ul 1 U/ul Human Placental RNase Inhibitor 1/ul 10 U/ul DNase I without RNase 5 ul 0.1 mol/L Tris-Cl, pH 8.3 5 ul 0.5 mol/L KCl 5 ul 15 mmol/L MgCl2 Add water to 50 ul incubate at 37°C for 30 min. 2. Add 50 ul of phenol/chloroform (3:1), vortex to mix, and centrifuge at maximum speed for 2 min to separate the phases. 3. Transfer the upper layer to a clean microcentrifuge tube, add 5 ul 3 mol/L ammonium acetate and 200 ul 100% ethanol. Place at 70°C for 30 min to precipitate the RNA. 4. High-speed centrifugation for 10 min. 4. Centrifuge at high speed for 10 min. remove the supernatant and wash the precipitate (precipitated RNA) with 500 ul of 70% ethanol. 5. 5. Dissolve the RNA precipitate in 20 ul of DEPC-treated water and measure the concentration of RNA accurately quantified by A260 using a spectrophotometer. 6. 6. Analyze 3 ug of RNA for differential display analysis by agarose/formaldehyde gel electrophoresis to check RNA integrity. The DNA-free RNA is stored at -80°C until analysis for differential display. 7. 7. For each RNA sample, label 4 microcentrifuge tubes as G, A, T, and C tubes corresponding to a dT primer. 8. 8. Dilute 1 ug of DNA-free RNA (Step 5) to 0.1 ug/ul and place on ice. 9. 9. Prepare a DNA-free total RNA or poly(A) + RNA reverse transcription reaction with 4 different simple anchoring primers (5' -T12MN-3 '; T12MG, T12MA, T12MT, T12MC, M is G, A or C). 4 ul of 5×MoMuMuRNA (Step 5) is used to prepare the reverse transcription reaction: 4 ul 5×MoMuLV reverse transcriptase buffer (final concentration 1×) 2 ul 0.1 mol/L DTT (final concentration 10 mmol/L) 1.6 ul 250 umol/L 4dNTP mixture (final concentration 250 mmol/L) 0.2 ug total RNA or 0.1 ug poly (A) + RNA 2 ul a 10 umol/L condensed anchored oligo(dT) primer ( T12MN; final concentration 1 umol/L) Adjust the volume of DEPC-treated water to 19 ul. 10. 10. incubate at 65℃ for 5 min to denature the secondary structure of mRNA, and continue to incubate at 37℃ for 10 min to denature the primer. 11. add 1 ul of 200 mRNA to each tube. 11. Add 1 ul of 200 U/ul of MoMuLV reverse transcriptase to each tube, mix well and incubate at 37℃ for 50 min. 12. 12. Heat at 95℃ for 5 min to inactivate the reverse transcriptase and collect the solution at the bottom of the tube by high-speed centrifugation. Place the tubes on ice and prepare the PCR immediately or store at -20°C for later use (stable for at least 6 months). 13. Prepare the following primers for each primer 13. Prepare 20 ul of reaction mix for each primer as follows: 10 ul H2O 2 ul10× amplification buffer (final concentration 1× ) 1.6 ul 25 umol/L 4dNTP mixture (final concentration 2 umol/L) 0.2 ul [ α-33P ] dATP 2 ul 12 umol/L of random decamer (final concentration 0.2 umol/L) 2 ul 10 umol/L dT primer ( T12MN; final concentration 1 mmol/L) 2 ul cDNA (12 steps) 0.25 U 5 U/ul Taq DNA Polymerase 14. Blow up and down to mix, cover with 25 ml of mineral oil. 15. 15. Amplify on a thermal cycler using the following program: 40 rounds: 30 s 94°C (denaturation) 2 min 40°C (denaturation) 30 s 72°C (extension) 1 round: 5 min 72°C (extension) Final step: 4 C (hold) 16. mix 3.5 ul of PCR product with 2 ul of formamide sample buffer and heat at 80 °C for 2 min. sample onto a 6% denaturing polyacrylamide gel. electrophoresis at 60 W for approximately 3 h until the xylene cyanide is within 10 cm of the bottom. 17. carefully remove a glass plate from the gel. 17. Carefully remove a glass plate from the gel. Place a piece of Whatman 3 MM filter paper over the gel, leaving no air bubbles between the gel and the filter paper. Without fixation in methanol/acetic acid, dry the gel for about 1 h at room temperature. 18. Use radioactive ink or a clock punch to mark the X-ray film and the dried gel shift to determine their orientation. Expose for 24-48 h at room temperature. 19. 19. Wash the slides, compare the gel slides to the gel, and mark the DNA bands of interest (bands that show up differently in different lanes) or mark the underside of the gel slides with a clean pencil or by cutting through the gel slides. 20. Cut out the gel block and the adherent Whatman 3 MM filter paper with a razor blade and place in a microcentrifuge tube. Add 100 ul of H2O and soak for 10 min at room temperature. 21. 21. Cover the tube and boil for 15 min. 22. 22. Centrifuge at high speed for two minutes to precipitate the gel mass and filter paper residue. Transfer the supernatant to a clean tube. 23. 23. To the supernatant, add 10 ul of 3 mol/L sodium acetate (final concentration 0.3 mol/L) and 5 ul of 10 mg/ml glycogen (as carrier). Add 400 ul of 100% ethanol and place at -70°C for 30 min. Centrifuge at 4°C for 10 min. 24. Wash the precipitate with 500 ul of 85% ethanol, allow to dry, and redissolve in 10 ul of H2O. 25. 25. Re-amplify 4 ul of eluted DNA in a 40 ul reaction volume, using the same primer profile and PCR conditions as in steps 13-15, except that 250 umol/L 4dNTP mixture (final concentration 20 umol/L) was added instead of 1.6 ul of 25 umol/L 4dNTP mixture, and no isotope was added. The remaining recovered DNA is stored at -20°C for future amplification (stable for several years). 26. 30 ul of each PCR sample was electrophoresed on a 1.5% agarose gel and stained with 0.5 ug/ml EB. The remaining PCR samples were stored at -20 °C (stable for several years). 27. Extract the desired amplified DNA band from the agarose gel. Use it as a probe for North-era blot analysis and screening of cDNA libraries. 28. 28. Subclone and sequence the remaining PCR samples for identification. Caveat Must be used by a person trained in the proper use of33P isotopes in an NRC-certified location. Preventing overexposure requires that isotope contamination precautions for personnel and instrumentation be implemented at all times. Common Problems 1. Material Human total cellular RNA or poly (A) + RNA 2. Reagents 1 U/ul Human Placenta RNase Inhibitor 10 U/ul DNase I (RNase-free) 0.1 mol/L Tris-Cl, pH 8.3 0.5 mol/L KCl 15 mmol/L MgCl2 3:1 (V/V) Phenol/Chloroform 3 mol/L sodium acetate, pH 5.2 100%, 70% and 85% ethanol DEPC-treated water Simplex-anchored oligo (dT) primer combinations (e.g. Genhunter) at 10 umol/L each: T12MG, T12MA, T12MT, T12MC (M for G, A or C) 5 × MoMuLV reverse transcriptase buffer 0.1 mol/L DTT 250 umol/L and 25 umol/L 4dNTP mixture 200 U/ul Moloney murine leukemia virus (MoMuLV) reverse transcriptase 10× PCR amplification buffer (using 15 mmol/L MgCl2, 0.1 mg/ml white gel; stored at -20°C) 10 uCi/ul [ α-33P ] dATP (>2000 Ci/mmol) 2 umol/L random decamer (e.g. GenHunter or Operon Technologies) 5 U/ul Taq DNA polymerase Mineral Oil Formamide Sampling Buffer 10 mg/ml of glycogen (no DNase) 3. Consumables 65°C, 95°C, 80°C and 100°C water baths Thermal cycler Whatman 3 MM Filter For more product details, please visit Aladdin Scientific website.
Human placenta RNase inhibitor DNase I Tris-Cl KCl MgCl2 3:1 (V V) Phenol Chloroform Sodium acetate Ethanol DEPC-treated water Simplex-anchored oligo (dT) Primer combinations MoMuLV Reverse Transcriptase Buffer DTT 4dNTP Mixture MoMuLV Reverse Transcriptase PCR Amplification Buffer [alpha-33P] dATP Random Decamer Taq DNA Polymerase Mineral Oil Formamide Sampling Buffer Glycogen (no DNase)
65°C, 95°C, 80°C and 100°C Water Bath Thermal Cycler Whatman 3 MM Filter Paper