Oneill Lab:IVT Template

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.
 * 2) *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.
 * 3) Determine the orientation of your insert by sequencing from the vector DNA.
 * 4) *It is critical to know the orientation of your insert relative to your cloning vector of choice.
 * 5) *This will allow you to choose the appropriate enzyme for IVT, and thus control which strand is synthesized.
 * 6) There are two methods for actually creating the template from this point.
 * 7) Typically the vector is linearized with a restriction endonuclease, such that the IVT enzyme will transcribe the insert without running onto the vector sequence.
 * 8) *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.
 * 9) Alternatively, PCR amplification off the vector can be used to create a linear product including the appropriate viral promoter site.
 * 10) *This is very easy when using plasmids such as pBluescript or the TA cloning vectors like pSC-a (from Stratagene) and TOPO.
 * 11) *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.
 * 12) *You will need to use the sequence derived orientation of your insert to determine which primers to use to transcribe the desired strand.
 * 13) The linear template must be cleaned up before use in IVT.
 * 14) *For digests, extract by PCI and ethanol precipitation.
 * 15) *Clean PCR products with Qiagen PCR Purification column. Add 20 &mu;g ProK and 2 volumes ReagentB. Incubate at 50&deg;C overnight. The following day extract with PCI and precipitation. Resuspend in 10 &mu;L of MilliQ water.