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    Introduction

    I am basing thing protocol on the protocol given by the pormega RiboMAX(TM) Large Scale RNA Production Systems—SP6 and T7 kit (link) Advices and thing to watch out for The Linear Control DNA supplied with each system contains a luciferase gene under the control of the appropriate SP6 or T7 RNA polymerase promoter. This DNA produces a transcript approximately 1,800 bases long. Since luciferase must be full-length to show activity, transcription and translation of the Control DNA followed by a luciferase assay is a convenient means to verify that full-length transcripts have been generated. Set up the appropriate reaction for SP6 or T7 RNA Polymerase at room temperature. Add the reaction components in the order shown, being careful to dissolve the DNA template in water before adding it to the reaction. For convenience, mix equal volumes of the 4 individual 100mM rNTPs provided to produce a solution that is 25mM for each nucleotide.

    Materials

    Procedure

    • PCR DNA Clean-up (monarch PCR and DNA cleanup kit) ononarch® PCR & DNA Cleanup KitMon
    • Master mix preparation
    1. Prepare the reaction mix
    Reaction mix
    Collapse table
    A
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    D
     
    		T7 Reaction Components Sample Reaction Control Reaction - positiveNegative control
     
    		T7 Transcription 5X Buffer 20µl 4µl4µl
     
    		rNTPs (25mM ATP, CTP, GTP, UTP) 30µl 6µl6µl
     
    		Linear DNA template (5-10µg total) 40µL1µL (control DNA) 1µL (control DNA)
     
    		plus nuclease free water plus 7µL (water)plus 7µL (water)
     
    		Enzyme Mix (T7) 10µL2µL0 µL
     
    		final volume100µL20µL18 µL
    1. Notes :
    • - DNA can precipitate in the presence of spermidine (a component of the Transcription 5X Buffer) if reactions are set up at colder temperatures.
    • - These reactions can be scaled up or down to suit your template requirements. A 1ml reaction will typically produce 2–5mg of RNA in 2–4 hours.
    1. Gently pipet the reaction to mix and incubate at 37°C for 2–4 hours.
    • Note: Do not freeze the completed transcription reaction. After the transcription reaction is complete, proceed directly to the DNase step or the removal of unincorporated nucleotides.
    • Because wa are doing miniprep before this step everything below is optional but I might be a good idea to do it anyway and put it on a gel to verify this step worked.
    • Removal of the DNA Template Following Transcription (note that this step can be skipped)
    1. Add RQ1 RNase-Free DNase to a concentration of 1u/µg of template DNA.
    1. Incubate for 15 minutes at 37°C.
    • Gel d'ARN --> voir si transcription ok

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