Oneill Lab:IVT

From OpenWetWare

Jump to: navigation, search

Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology

Back to O'Neill Lab Protocols

This is the protocol for Ambion's MAXIscript IVT kit.

Contents

In Vitro Trascription

  1. Thaw transcription buffer and nucleotides. Vortex nucleotides and keep on ice. Keep transcription buffer at room temp.
  2. Assemble reaction at room temperature.
    • Spermidine in the transcription buffer can coprecipitate the template DNA if assembled on ice.
    • Do not add the transcription buffer until both water and template DNA are in the reaction tube.
  3. Incubate reactions at 37°C for 10 minutes if radiolabelling or for 1 hour if using nonisotopic labels or unlabeled nucleotides.
  4. Add 1 μL TURBO DNase 1
  5. Incubate at 37°C for 15 minutes
  6. Add 1 μL of 0.5M EDTA
In vitro Transcription
Component Isotopic Unlabelled Nonisotopic
Nuclease-free water to 20 μL to 20 μL to 20 μL
DNA template 1 μg 1 μg 1 μg
10x Transcription Buffer 2 μL 2 μL 2 μL
10 mM ATP/CTP/GTP 1 μL each 1 μL each 1 μL each
10 mM UTP -- 1 μL 0.6 μL
Labeled UTP 5 μL -- 1.2 μL
Enzyme Mix 2 μL 2 μL 2 μL

Cleaning up IVT product

Following transcription it may be necessary to hydrolyze your probe or remove free nucleotides. For RNase protection assays or other experiments requiring only the full length probe, it is necessary to gel purify the IVT product. To remove unincorporated nucleotides proceed directly from DNase treatment to one of the following precipitation.

Ammonium Acetate Precipitation

  1. Bring reaction volume up to 50 μL
  2. Add 5 μL of 5M Ammonium Acetate and vortex
  3. Add 3 volumes of 100% ethanol (RNase free).
  4. Chill at -20°C for at least 30 minutes.
  5. Spin for at least 15 minutes at max speed at 4°C
  6. Remove supernatant and wash once with 70% ethanol.
  7. Resuspend pellet in 50 μL of nuclease free water.
  8. Aliquot and store at -80°C
    • There is no need to aliquot isotopically labeled transcripts since they degrade from radiolysis in 2-4 days

Lithium Chloride Precipitation

  1. Bring reaction volume to 40 μL
  2. Add 5 μL of 4M LiCl
  3. Add 140 μL 100% ethanol
  4. Precipiate at -80°C for 1 hour
  5. Spin for 20 minutes at max speed at 4°C
  6. Remove supernatant and wash once with 70% ethanol.
  7. Resuspend pellet in 96 μL DEPC treated water + 3 μL RNAsin + 1 μL 100mM DTT.
  8. Aliquot and store at -80°C
Personal tools