gRNA一步法简便合成方案 T7启动子

R:AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACF:TAATACGACTCACTATAGTTTTAGAGCTAGAAATAGC1. Denature at 98C for 2 minutes 2. Anneal at 50C 10 minute 3.Extend at 72C 10 minutes 4. Run 2.5% Agarose Gel, you should see a 120 bp band when 3-5卩1 loaded on gelStep 3 In vitro Transcription using assembled oligo as templateUse 2-3 卩1 assembled oligos and proceeds for IVT reaction using T7 quickHigh yield RNA synthesis kit (NEB) Reaction:2X NTP mix10卩lAssembled oligos 3卩l T7 polymerase1卩lWater6卩l Incubate at 37C for 2-16 hoursAdd 1卩l DNAse (RNAse free DNAse from the kit) and incubate at 37C for 15 minutes.A. Purify RNA using isopropanol/sodium acetate Add 75 gl water to the reaction Add equal volume of isopropanol (100 gl) Add 1/10 volume of sodium acetate or ammonium acetate (pH5.2) Chill at -20C for 30 minutes Precipitate RNA by spinning at maximum speed for 15 minute at 4C Wash pellet with 70% ethanol (200 gl) Spin at maximum speed for 5 minutes and dry pelletDissolve pellet in 20 pl water