BioArray™ HighYield™ RNA Transcript Labeling Kit (T7)

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Precipitate in the reaction buffer or DTT

• After many freeze-thaw cycles, a precipitate may form. If the precipitate does not solubilize after gentle mixing, do not use.
  
  

Low Yield

• The most likely cause of low yield in a transcription reaction is poor quality or impure template. Carry-over of phenol will inhibit the reaction. To remove phenol, wash the template twice with 70% or 80% ethanol. The presence of excess T7 promoter containing primers during synthesis of cDNA template can also decrease yield. Following synthesis of the cDNA template the primers can be removed by precipitating the cDNA with 2.5M ammonium acetate and 2.5 volumes of absolute ethanol. The precipitate should be spun immediately at room temperature for 20 minutes. If other salts are used or if the sample is frozen the primers may also precipitate resulting in their incomplete removal. If interference by excess primers persists, the starting concentration of primers can be reduced in cDNA synthesis reaction. This is recommended when starting with reduced amounts of RNA.
  Alternatively, cDNA can be purified using Enzo’s cDNA Purification Kit (ENZ-42407)
• Some cDNA synthesis reactions may produce cDNA that has been primed with RNA instead of the T7 promoter-containing oligo primer. This is more likely to occur when starting with low quality RNA. The RNA-primed cDNA contains no T7 promoter sequence and thus will not be transcribed.