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DNA Vector-Based shRNA-Expression Systems 2353.3.3. Cloning of the Gene-Specific shRNA-Expression Vectors1. Mix 2 µL of ClaI/HindIII-digested vectors and 8 µL of annealed shRNA-codingDNA templates in a 1.5-mL Eppendorf tube in a reaction with 2 µL of 10X ligationbuffer and distilled H 2O to total 19 µL (see Note 8).2. Add 1 µL of T4 DNA ligase.3. Incubate in 16°C water bath overnight.4. Transform 200 µL of XL 1-blue competent cells with 20 µL of ligated mixtures.5. Plate on LB agar plates containing 100 µg/mL of ampicillin.6. Incubate in 37°C incubator overnight.3.3.4. Screening of the shRNA-Expression Template Positive Clones1. Inoculate four selected colonies into 3 mL LB broth containing 100 µg/mL ofampicillin (see Note 9).2. Incubate in 37°C incubator overnight.3. Purify plasmid DNAs from 1.5 mL overnight culture by using plasmid mini purificationkit, and elute the plasmid DNAs with 50 µL of TE (pH 8.0).4. Check isolated plasmid DNAs by single digestion with restriction enzyme ClaI orHindIII. Digest 2 µL of purified plasmid DNA in a 1.5-mL Eppendorf tube in areaction with 2 µL of 10X restriction enzyme buffer, 2 U of ClaI or HindIII, anddistilled H 2O to total 20 µL in 37°C water bath for 1 h.5. Analyze 10 µL of digested DNAs on a 0.8% (w/v) agarose gel with an appropriatemolecular weight marker. The positive shRNA-expression clones containingrestriction enzyme HindIII site but usually losing restriction enzyme ClaI site aredigested only with HindIII and not digested with ClaI. Plasmids showing thisrestriction enzyme-digestion pattern are presumably correct and should be confirmedby directly sequencing.3.3.5. Sequencing of the shRNA-Expression TemplatesPlasmid DNA is sequenced by using an automated DNA sequencer, whichuses the dideoxy sequencing method with fluorescent dyes.1. Set up cycle sequencing reaction: 500 ng plasmid DNA, 3.2 pmol of T7 or SP6promoter primer, 8 µL ABI Prism dGTP BigDye terminator, and distilled H 2O tototal 20 µL.2. Perform the PCR reaction by using the following thermocycling parameters:Step Time Temperature (°C) CyclesInitial denaturation 2 min 94 1Denaturation 30 s 96 –Annealing 15 s 50 25Extension 4 min 60 –3. Amplify plasmid DNA containing the correct sequence by using plasmid maxipurification kit, and elute the plasmid DNAs with 500 µL of TE (pH 8.0).