Synthesis of ARCA capped mRNA

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Linearized plasmid DNA and PCR products that contain a T7 RNA polymerase promoter sitecan be used as templates for in vitro transcription with mMESSAGE mMACHINE T7 Ultra. Ingeneral, any DNA with a T7 promoter site, that is pure enough to be easily digested withrestriction enzymes can be used for in vitro transcription.Equipment preparationLab bench and pipettorsBefore working with RNA, it is always a good idea to clean the lab bench and pipettors withan RNase decontamination solution (e.g. RnaseAWAY, Invitrogen).Gloves and RNase-free techniqueWear laboratory gloves at all times during this procedure and change them frequently. Theywill protect you from the reagents, and they will protect the RNA from nucleases that arepresent on the skin. Use RNase-free pipette tips (filter tips) to handle all reagents and mRNA.Transcription reaction assembly‣ Thaw the frozen reagents. Place the RNA Polymerase Enzyme Mix on ice, it is stored inglycerol and will not be frozen at –20°C. Vortex the 10X T7 Reaction Buffer and the T7 2XNTP/ARCA until they are completely in solution. Once thawed, store the ribonucleotidesT7 (2X NTP/ARCA) on ice, but keep the 10X T7 Reaction Buffer at room temperaturewhile assembling the reaction.All reagents should be microfuged briefly before opening to prevent loss and/orcontamination of material that may be present around the rim of the tube.‣ Assemble transcription reaction at room temperature (the spermidine in the 10X T7Reaction Buffer can coprecipitate the template DNA if the reaction is assembled on ice).Add the 10X T7 Reaction Buffer after the water and the ribonucleotides are already inthe tube.For transcription of pGEM-GFP-64A, add:ComponentAmountNuclease-free water To 20 µlT7 2x NTP/ARCA 10 µl10x T7 reaction buffer 2 µlLinearized plasmid template 1 µgT7 enzyme mix 2 µlFor transcription of control plasmid (pTRI-Xef 1), add:ComponentAmountNuclease-free water 5 µlT7 2x NTP/ARCA 10 µl10x T7 reaction buffer 2 µlControl plasmid (pTRI-Xef 1) 1 µlT7 enzyme mix 2 µl
‣ Pipette the mixture up and down gently, and then microfuge tube briefly to collect thereaction mixture at the bottom of the tube.‣ Incubate at 37°C, for 2 hours.‣ Add 1 µl TURBO DNase and mix well. Incubate at 37°C for 15 minutes.‣ Inactivate RNA polymerase and DNase by adding 2 µl 0.5M EDTA (pH 8.0) and incubateat 65°C for 10 minutes.
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Synthesis of ARCA capped mRNA

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