µMACS One-step T7 Template Kit

µMACS One-step T7 Template Kit

The µMACS One-step T7 Template Kit facilitates the in-column synthesis of double-stranded cDNA, containing a T7 promotor, from limited biological sample, including biopsies or low numbers of cells. A subsequent in vitro transcription (IVT) can be performed in the same column, thus minimizing loss of sample and concurrently allowing for an efficient amplification of antisense-RNA (aRNA) for microarray gene expression analysis.

Detailed procedure

After cell lysis and clearing of the cell lysate, the mRNA is magnetically labeled with µMACS MicroBeads, containing an attached Oligo(dT)-T7 nucleotide sequence. Due to the small size of the MicroBeads, the hybridization to the poly(A)
+
tail of the mRNA molecules is completed within seconds. The sample is loaded onto a µ Column placed in the magnetic field of the thermoMACS Separator. After washing, the magnetically labeled mRNA is retained on the column. A ready-to-use reverse transcriptase enzyme mix is added onto the column and the thermoMACS Separator is set to 42 °C, enabling efficient first-strand cDNA synthesis. Subsequently, a second strand synthesis mix containing DNA polymerase, DNA ligase, and RNase H generates double-stranded cDNA. After thorough washing, pure double-stranded cDNA is eluted from the column.
Optionally, before elution of the cDNA, the antisense RNA can be synthesized on the same column within three hours by adding a T7 RNA polymerase (enzyme is not included in the kit). In this case, the amplified antisense RNA is eluted, purified, and ready for further applications, such as labeling and microarray hybridization.
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