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===Maxiscript (Ambion) T7 Protocol=== DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification. 1. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) on ice, but keep 10X transcription buffer at room temp 2. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!! bring to 20 uL with Nuclease free water X uL Nuclease free H2O X uL DNA template (list 1 ug) 2 uL 10X Transcription Buffer 1 uL 10 mM ATP 1 uL 10 mM CTP 1 uL 10 mM GTP 1 uL 10 mM UTP 2 uL T7 Enzyme Mix 3. Incubate reactions at 37C overnight. (>10 uM limiting nucleotide) 4. (Optional) Add 1 uL of TURBO DNase, mix well, incubate at 37C for 15 minutes. 5. (Optional) Add 1uL of 0.5 M EDTA to stop the reaction 6. Remove unincorporated nucleotides for quantitation. (ethanol precipitation or column precipitation). Can also run TBU gel to view sample and controls Ethanol preciptation: 1. Add 30 uL water to DNase-I treated transcription reaction to bring the volume to 50 uL. 2. Add 3 volumes 10% EtOH, .1 volumes 3M NaOAc, and 1/300 volumes glycol Blue to the solution obtained. In this case: 5 uL NaOAc (mix after adding) 150 uL 100% EtOH 1 uL (1/10 dilution Glycol Blue) 3. Chill solution in -80 for 30 minutes, cool centrifuge to 4C 4. Spin at 4C for >15 minutes at max speed. Chill 75% EtOH. 5. Blue pellet should be visible, Discard supernatent. 6. Wash the pellet with cold 75% EtOH 7. To completely remove incorporated NTPs, resuspend the pellet in 50 uL nuclease free water and repeat steps 1-6. Otherwise resuspend with nuclease free water. 8. Store RNA at -80, in aliquots if desired. Aliquot in use can be at -20. *Now can nanodrop for quanitation or run gel. fragment RNA, polyA tail addition, adapter addition to 5' and 3' ends.
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