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œœœœœ@T7 run-off transcription@œœœœœ



RNA can be synthesized in vitro using RNA polymerase from T7 or SP6, if the template DNA is cloned adjacent to the appropriate promoter sequence. Such cloning vecotrs, i.e. pSP, pGEM and pTZ plasmids, are commonly and commercially available. In vitro synthesized RNAs are useful because of the ability to prepare ug quantities of the RNA from all other nucleic acids & proteins, and because the RNA can be designed to the users specifications.
The plasmid to be transcribed must first be linearized at a convenient restriction site so that the RNAP "runs-off" the end of the template, yeilding a discrete transcript.

  1. Materials

    1. 5X PO4/DTT(1 ml)
      100mM NaPO4 (100ul of a 1M solution pH 7.7)
      50 mM DTT (50 ul of a 1 M stock )
      (850 ul ddH2O)

    2. 5X Magnesium/Spermidine (1 ml)
      40 mM MgCl2 (40 ul 1 M stock)
      20 mM Spermidine (100 ul 200 mM stock)
      (860 ul ddH2O)

    3. 10X NTP Stock (500 ul)
      100 ul 50 mM Tris-HCl pH 8.0
      100 ul 20 mM ATP
      100 ul 20 mM CTP
      100 ul 20 mM GTP

    4. 10X UTP Stock (100 ul)
      2 ul 20 mM UTP
      98 ul 50 mM Tris-HCl pH 8.0

    5. 1X TE, 0.1% SDS (1L)
      10 mM Tris (10 ml 1M Tris-HCl pH 8.0)
      1 mM EDTA (2 ml 0.5M EDTA pH 8.0)
      0.1% SDS (10 ml 10% SDS)

    6. Column dye (per 20ml)
      Final Conc.
      0.25% bromophenol blue 50 mg
      50% glycerol 10ml
      1/2 X TE 10ml 1X

    7. Elution buffer
      0.5M NH4OAc
      10mM Mg(OAc)2
      1mM EDTA
      0.1% SDS

    8. Sequencing dye
      0.25% bromophenol blue
      0.25% xylene cyanol FF
      50% urea
      1X TBE

  2. Methods

    1. Working on ice, mix:
      4 ul 5X Phosphate/DTT buffer
      4 ul 5X Magnesium/Spermidine Buffer
      2 ul 5 mM NTP's (A,C,G)
      2 ul 1 mM UTP (cold)
      2 ug Linear Template
      5 ul alpha P32 UTP* (10 uCi/ul)
      1 ul T7 Polymerase (40 u/ul)
      20 ul Add ddH2O to
      - Alpha P32 GTP may also be used with 5mM NTPs (A,U,C) and 1 mM GTP.
      - The amount of radionucleotide may be increased to allow for decay of the 32P.
      - For cold RNA synthesis, omit the radionucleotide and replace the nucleotides with 4ul of a 5mM GTP/UTP/ATP/CTP mix.

    2. Incubate the reaction for 3 hours at 37‘.

    3. G-50 column purification (for labeled RNA probes)

      *** Since the probe is an RNA probe, An effort should be made to reduce the amount of RNase contamination.***

      1. DEPC treat an 8" x 3/8"dia. Econo column and a small section of tubing. This is done by putting 200ul dep in the bottom of a 250 ml graduated cylinder and filling it with EtOH. Then drop in your tube and column. Allow this to set for >10 min.
      2. Start to degas some of the hydrated Sephadex G-50.
      3. Drain the column and rinse well with 1XTE,0.1%SDS.
      4. Put the tube on the end and clamp off the tube, the column should be mounted onto a ring stand.
      5. After the Sephadex is done degassing, fill the column with the suspended Sephadex and open the clamp at the bottom of the column. Use a screw capped 50 ml dispo tube to catch the buffer. Add more Sephadex as the solution drains. Continue this until the Sephadex is about an inch from the top of the glass.

    4. Column chromatography

      1. Add 50ul TE and 10ul column dye to the sample.
      2. Apply the sample onto the G50 sephadex column.
      3. Run the column with TE-SDS, collecting the first 20 fractions, 12 drops each. Wash the column in TE-SDS until the blue dye is all washed out of the column.
      4. Spot 2ul of each fraction onto 4-5mm squares of 3MM paper. Count each square in 4ml scintillation fluid.
      5. Pool the G50 excluded peak (i.e. the first peak), which usually is at fractions 7-10. Store at -20C until use.

    5. Gel purification (for unlabeled RNAs or substrates)

      1. Mix the sample with an equal volume of seq dye & incubate 2min at 65C.
      2. Load the sample onto a 1mm thick 6% sequencing-recipe gel & electrophorese 500-1000 volts until the bromophenol blue reaches the bottom of the gel (adjust, as required by the length of the RNA).
      3. Place the gel onto a saran-wrap covered PEI plate & examine under UV light. Use a razorblade to exice the transcript bands from the gel.
      4. Soak each band in 1ml elution buffer ON at 4C.
      5. Precipitate the RNA with 2ml EtOH at -70C (dry-ice:EtOH bath) for 5min., then centrifuge 5 min, 4C to pellet. Drain the pellet & rinse with -20C 70% EtOH, drain & dry in a rotovap.
      6. Resuspend the RNA in 50-100ul ddH2O. Determine the concentration of RNA in the sample using the Ethidium bromide spot test.

PaceLabs procedure

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