Differential reduction cDNA library creation experiment
Differential reduction cDNA library creation experiment
Differential subtraction libraries contain mRNA cDNA clones that are present in one cell or tissue but not in another. This cDNA library is used to isolate a set of cDNA clones corresponding to a class of mRNAs or to isolate cDNA clones of a specific mRNA. The process of screening cDNA clones is laborious. Source: The Compact Guide to Molecular Biology, Fifth Edition.
Operation method
basic program
Materials and Instruments
[+] and [-] cDNA libraries (ATCC or Stratagene) Move Materials, reagents and consumables required for the experiment are described under "Others". 1. Obtain cDNA libraries from [+] and [-] cells or tissues. This protocol assumes that the [+] and [-] libraries are λ phage libraries. If the vector for both libraries is a plasmid, then they both require only 100 ug. The insert fragments need to be purified by agarose gel electrophoresis, not just by sucrose gradient centrifugation. 2. 2. purify >1 mg of DNA from each of the [+] and [-] libraries using a large-scale DNA purification column, and resuspend in TE buffer at 1 mg/ml. 3. 3. Digest 1 mg of DNA from each of the two libraries in a 1.5 ml microcentrifuge tube as follows (final volume 1.167 ml): 1 ml library DNA (1 mg) 0.117 ml 10×EcoRI Buffer 0.05 ml EcoRI (1000 U) Mix well and incubate at 37°C for 5 h. Add 40 ul of 0.5 mol/L EDTA, pH 8.0 and incubate at 65°C for 10 min to stop the reaction. During the digestion time, four 10%-40% sucrose gradient solutions were prepared in 38 ml SW-28 tubes. Two tubes are labeled [+] and two tubes are labeled [-]. The EcoRI site inherent in the inserted cDNA fragment will also be cut. The partial-length cDNA clone obtained in this step can be used to make a probe for screening full-length clones from the starting [+] library, if this is the case. Newer cloning vectors containing sites such as NotI (e.g., λZAP) are now available to overcome these difficulties almost completely. 4. 4. Add an equal volume of 10% sucrose solution to each digestion product and mix well. The mixed digestion product of the [+] library is divided into two equal portions and carefully added to two tubes of the 10% to 40% sucrose gradient labeled [+]. Similarly, the DNA digest of the [-] library is added to the sucrose gradient solution labeled [-]. The sucrose gradient is centrifuged overnight (18-24 h) at 20°C, 122,000 g (26,000 r/min, SW-28 rotor). 5. Remove the DNA from the [-] library carefully with a pipette. 5. Carefully remove 0.2 ml of the component from the bottom of the tube with a pipette tip, and place each of the remaining components in a labeled microcentrifuge tube and store at 4°C. 6. 6. Take 20 ul of each other fraction and analyze the inserted DNA fragments on a 1.5% agarose gel prepared in TBE buffer. 7. Add 0.3 ml TE buffer and 1 ml 95% ethanol to each tube, mix well, and precipitate the inserted DNA fragments by placing the tube at -20°C for 2 h or on dry ice for 15 min. Sufficient NaCl to precipitate the DNA is already present in the fraction, and the sucrose in the sucrose gradient fraction must be diluted in order to precipitate the DNA. For low-density fractions, a 1- to 3-fold dilution is often required. Higher dilutions are required for efficient DNA recovery in the high-density fractions. 8. 8. After thawing the precipitate solution, collect the DNA by centrifugation at high speed in a microcentrifuge tube for 15 min. aspirate and save the supernatant until the recovered DNA has been checked and confirmed before discarding. Add 0.5 ml of 70% ethanol to each tube, centrifuge again, aspirate the supernatant, and air-dry the precipitate. 9. 9. Resuspend and incorporate insert DNA from the ［+］ library in TE buffer at a final concentration of 0.2 mg/ml. Store at -20℃. 10. Resuspend and combine insert DNA from the ［-］ library in 100 TE buffer on ice. Keep a small 400 ng portion of each of the ［+］ and ［-］ cDNAs separately for evaluation of the final library. From 1 mg of total library DNA, it is expected that greater than 10-15 insert DNA can be recovered. small portions of ［+］ and ［-］ DNA can also be radiolabeled and used as differential screening probes for the ［+］ library. 11. Mix the reaction system as follows 11. Mix the reaction system (final volume 112 ul) in the following order, removing the EcoRI end of the ［-］ DNA. 100 ul ［-］ insert DNA (10-15 ug) 11 ul 10 × S1 Nuclease Buffer 1 ul 1:500 S1 nuclease (2 U) Vortex, centrifuge to the bottom of the tube, and incubate at 37°C for 30 min. 12. Add the following reagents to terminate the procedure 12. Add the following reagents to terminate the reaction: 5 ul 0.5 mol/L EDTA, pH 8.0 200 ul TE buffer 300 ul Phenol/Chloroform/Isopropanol Vortex, centrifuge for 1 min to separate the phases and transfer the upper aqueous phase to a new centrifuge tube. Add 30 ul 3 mol/L sodium acetate, pH 5.2, and 700 ul ethanol. Freeze and collect the DNA by centrifugation as in steps 7 and 8. Resuspend the precipitate in 100 ul TE buffer. 13. 13. Digest the S1 nuclease-treated ［-］ insert DNA into small fragments using AluI and RsaI. Mix the reaction system in the following order (final volume 121 ul): 100 ul ［-］ insert DNA (10-15 ug) 12 ul 10× AluI buffer 5 ul AluI (50 U) 4 ul RsaI (60 U) Vortex and mix well, centrifuge to the bottom of the tube and incubate at 37°C for 3 h. Add 5 ml of 0.5 mol/L EDTA, pH 8.0 and incubate at 65°C for 10 min to stop the reaction. Remove 5 ml of digested product for electrophoresis. 14. 14. Add 200 ul TE buffer and 300 ul phenol/chloroform/isopropanol; extract according to step 12, ethanol precipitation. Resuspend in TE buffer at 1 ug/pl. 15. 15. Check 5 ul of digested product from step 13 by electrophoresis on a 2% agarose gel (in TBE buffer), EB staining. ［The digested fragment of [-] DNA should be between 50 and 200 bp. 16. 16. Hybridize the [+] insert DNA with the [-] DNA fragments by adding the following reaction system (final volume 51 ul) to a 0.4 ml microcentrifuge tube 25 ul deionized formamide (50% final volume) 10 ul [-] DNA fragment (10 ug) 1 ul [+] insert DNA (0.2 ug) 12.5 ul 20×SSC (5× final concentration) 0.05 ul 1 mol/L NaPO4, pH 7.0 (10 mmol/L final concentration) 0.5 ul 0.1 mol/L EDTA, pH 8.0 (1 mmol/L final concentration) 0.5 ul 10% SDS (0.1% final) 1.0 ul 10 mg/ml yeast tRNA (0.2 mg/ml final concentration) Vortex and mix well, centrifuge to the bottom of the tube, denature in boiling water bath for 5 min, gently shake the tube again, and incubate at 37℃ for 18-24 h. 17. Add 200 ul TE buffer solution, and then add the yeast tRNA. 17. Add 200 ul TE buffer and transfer the mixture to a 1.5 ml microcentrifuge tube. Wash the hybridization tube with 250 ul of TE buffer and add the hybridization mixture (now 500 ul ) Add 500 ul of phenol/chloroform/isopropanol, vortex, and centrifuge for 1 min to separate the phases. 18. Transfer the upper aqueous phase to the upper aqueous phase. 18. Transfer the supernatant phase to a new centrifuge tube. Extract again with 500 ul phenol/chloroform/isopropanol as in the previous step. Add 50 ul 3 mol/L sodium acetate, pH 5.2, 1 ml ethanol and precipitate according to steps 7 and 8. The precipitate was resuspended in 12 ul TE buffer. 19. Connect the insert DNA to the λgt10 (not λgt11) phage arm. Mix the following reaction system (final volume 25 ul): 12 ul Insert DNA 10 ul λgt10 phosphorylated arm (10 ug) 2.5 ul 10× Ligation Buffer 0.5 ul T4 DNA Ligase (200 U) Blow gently up and down with a pipette and incubate overnight at 12-15°C. 20. Prepare a fresh E. coli. 20. Prepare a fresh overnight culture of E.coli C600hflA. The next morning, package the ligation product from step 19 with 8-10 commercial λ phage packaging extracts according to the manufacturer's instructions. The λgt10 vector is used here because it is selective for recombinants when grown in a suitable host bacterium. 10 ug of the λ phage vector corresponds approximately in moles to the number of moles of the EcoRI end of the incorporated ［+］ DNA. The EcoRI end of the [+] DNA must be taken into account, even if only a small fraction of the [+] inserted DNA fragments can be cloned after denaturing and hybridization. The recommended 10 ug of vector and not less than 8-10 packaging extracts ensure good library diversity. 21. 21. Add the suspension medium to the packaging mixture and pour into a 5 ml polypropylene tube, adjusting the final volume to 2 ml. Add 2 drops of chloroform and shake by hand for 3 s to allow the chloroform to settle. 22. 22. According to the amplification protocol of the library, mix 0.2 ml with 3 ml of fresh C600hfA solution and inoculate a total of 10 plates of 150 mm. The plates were incubated at 37℃ overnight. 23. 23. In the morning of the next day, take one plate and count the phage spots, multiply the number by 10 to get the total number of recombinants in the library. 24. 24. Elute the phage on the plate with a suspension medium or skip the individual phage spots for screening. 25. Evaluate the newly created differential subtraction library. The best way to do this is to amplify the library and differentially screen two nylon membranes from a single 150 mm plate with 20,000 to 40,000 recombinants. One is hybridized to the total [+] cDNA probe and the other to the total [-] cDNA probe. The total [+] and [-] cDNA probes are the [+] and [-] cDNAs that were preserved in step 10 of the radiolabeling procedure. most of the clones should hybridize to the [+] probe, and almost none to the [-] probe. Hybridization with a protein probe such as actin or tubulin is inappropriate, and based on a variety of factors, it is assumed that no (or very little) hybridization occurs. Common Problems 1. Materials [+] and [-] cDNA libraries (ATCC or Stratagene) 2. Reagents TE buffer EcoRI and 10×Ec0RI Buffer 0.5 mol/L EDTA, pH 8.0 10% (m/V) Sucrose Solution 1.5% and 2% agarose gels TBE buffer 95% and 70% ethanol S1 Nuclease (Sigma) 10×S1 Nuclease Buffer 25:24:1 (V/V/V/V) phenol/chloroform/isopropanol 3 mol/L sodium acetate, pH 5.2 AluI and 10×AluI buffer RsaI Deionized methyl uglutamine (Fluka, IBI or American Bioanalytical) 20×SSC 1 mol/L NaPO4, pH 7.0 10% (m/V) SDS 10 mg/ml yeast tRNA 24 : 1 (V/V) chloroform/isopropanol Phosphorylated λgt10 linker arm (Stratagene) 10× T4 DNA ligase buffer T4 DNA ligase (in sticky end units; New England Biolabs) E.coli C600flA λ Phage packaging extract (Stratagene) Suspension medium (SM) 3. Consumables SW-28 rotor and 38 ml centrifuge tubes (Beckman) or other similar tubes 0.4 ml microcentrifuge tubes For more product details, please visit Aladdin Scientific website.
TE buffer EcoRI EDTA Sucrose solution Agarose gel TBE buffer Ethanol S1 nuclease S1 nuclease buffer Phenol Chloroform Isopropanol Sodium acetate AluI RsaI Deionized methyl violet amine SDS Yeast tRNA Chloroform Isopropanol Phosphorylated λgt10 ligand arm T4 DNA ligase E.coli C600flA λ Phage Packaging Extract Suspension medium
Centrifuge tubes