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The Ambion® Cells-to-cDNA™ II Kit (patent pending) produces cDNA from cultured mammalian cells in less than 2 hr. No RNA isolation is required. This kit contains sufficient reagents for 40 reactions and is ideal for those who want to perform reverse transcription reactions on small numbers of cells, numerous cell samples, or for scientists who are unfamiliar with RNA isolation.
• No RNA purification required • From cells in culture to cDNA in less than 2 hr • Detect rare messages in as few as 1 cell • Ideal for labs not equipped for RNA isolation
Ideal for RT-PCR Applications The Cells-to-cDNA™ II Kit (patent pending) integrates RNase inactivation and DNase I treatment into an RT-PCR compatible cell lysis buffer, eliminating RNA isolation altogether. Most cell lysis buffers contain strong denaturants that if carried over into enzymatic reactions would inhibit or inactivate most enzymes. If a lysis buffer without strong denaturants is used, endogenous RNase can quickly degrade cellular RNA. The Cells-to-cDNA™ II Kit contains a novel Cell Lysis Buffer that inactivates endogenous RNases without compromising downstream enzymatic reactions. After inactivation of RNases, the cell lysate can be directly added to a cDNA synthesis reaction. Cells-to-cDNA™ II is compatible with both one-step and two-step RT-PCR protocols.
Quantitative—Linear Results and No Detectable DNA Contamination To illustrate the quantitative, linear response of Cells-to-cDNA™ II to variations in input cell number, a real-time quantitative RT-PCR experiment was performed using the ABI 7700 Sequence Detection System. Plotting cycle threshold (Ct) versus cell concentration for GAPDH yielded a standard curve with a correlation of 0.99. Thus, Cells-to-cDNA™ II yields linear results for 1 to 10,000 cells/ µLin a real-time RT-PCR two-step assay. It should also be noted that the minus-template PCR control for the GAPDH experiment was negative, indicating complete removal of genomic DNA.
Simple Procedure The kit comes with everything you need to go from cultured cells to PCR-ready cDNA in less than 2 hr; no RNA isolation is required. Cells are first washed with PBS and then resuspended in the Cell Lysis Buffer. A brief heating simultaneously lyses the cells and inactivates RNases. An optional DNase digestion is then performed to degrade genomic DNA, followed by a heat inactivation step. A fraction of the cell lysate is then used in a reverse transcription reaction with the included reagents.
Accessory Products: SuperTaq™ Thermostable Taq DNA Polymerase is recommended and available separately (SKU#s AM2050 and AM2052). For optimal amplification of fragments greater than 1 kb, use SuperTaq™ Plus Thermostable Taq DNA Polymerase (SKU#s AM2054 and AM2056).
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