Oneill Lab:IVT Template

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

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Good hybridization using riboprobes requires a high purity, well designed template for in vitro transcription. This protocol provides a straightforward strategy for producing a template utilizing TA cloning and PCR amplification.

Generating the Template

  1. Clone the CDS or region of interest for your probe into a plasmid vector.
    • The simplest method for this is PCR amplification off cDNA followed by TA cloning. If this is not possible, traditional digestion and subcloning may be required.
  2. Determine the orientation of your insert by sequencing from the vector DNA.
    • It is critical to know the orientation of your insert relative to your cloning vector of choice.
    • This will allow you to choose the appropriate enzyme for IVT, and thus control which strand is synthesized.
  3. There are two methods for actually creating the template from this point.
    1. Typically the vector is linearized with a restriction endonuclease, such that the IVT enzyme will transcribe the insert without running onto the vector sequence.
      • This can become difficult for situations where no unique restriction site is available for use, or for very large inserts where complete transcripts may be less abundant.
    2. Alternatively, PCR amplification off the vector can be used to create a linear product including the appropriate viral promoter site.
      • This is very easy when using plasmids such as pBluescript or the TA cloning vectors like pSC-a (from Stratagene) and TOPO.
      • Utilize the M13 primer binding site flanking the viral promoter you wish to use in addition to a gene specific oligo and you will amplify a linear dsDNA molecule containing your entire insert plus the promoter you wish to use.
      • You will need to use the sequence derived orientation of your insert to determine which primers to use to transcribe the desired strand.
    3. The linear template must be cleaned up before use in IVT.
      • For digests, extract by PCI and ethanol precipitation.
      • Clean PCR products with Qiagen PCR Purification column. Add 20 μg ProK and 2 volumes ReagentB. Incubate at 50°C overnight. The following day extract with PCI and precipitation. Resuspend in 10 μL of MilliQ water.