Invitrogen™ FirstChoice™ RLM-RACE Kit

Description

FastAP™ Thermosensitive Alkaline Phosphatase, 10X FastAP Buffer, Tobacco Acid Pyrophosphatase, 10X TAP Buffer, T4 RNA Ligase, 10X T4 RNA Ligase Buffer, M-MLV Reverse Transcriptase, RNase Inhibitor, 10X RT Buffer, 2.5 mM dNTP Mix, Mouse Thymus RNA (1 mg/ml), Random Decamers, 5' RACE Adapter, 3' RACE Adapter, 5' RACE Outer Primer, 5' RACE Inner Primer, 3' RACE Outer Primer, 3' RACE Inner Primer, 5' RACE Outer Control Primer, 5' RACE Inner Control Primer, 5' PCR Control Primer, 3' RACE Control Primer and Ammonium Acetate Stop Solution should be stored at -20°C. Nuclease-free Water may be stored at any temperature.

The FirstChoice™ RLM-RACE Kit is designed to amplify cDNA from full-length, capped mRNA through RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE). The kit ensures the amplification of only full-length transcripts by eliminating ribosomal RNA, fragmented mRNA, tRNA, and contaminating genomic DNA during the amplification process. This kit is a major improvement over the basic rapid amplification of cDNA ends (RACE) protocol.

Note: calf intestinal phosphatase (CIP) had been replaced with FastAP Thermosensitive Alkaline Phosphatase adjusted protocol eliminate the need to use phenol/chloroform extraction and ethanol precipitation. If chosen, phenol/chloroform step can be used with updated kit after verifying reaction volumes.

Features of the FirstChoice RLM-RACE Kit include:

• Selects 5' and/or 3' ends of full-length, capped mRNA
• Optimized to ensure detection of even the rare mRNA, yielding specific product
• Allows to finish experiment in less than a day

Selects full-length mRNA—no rRNA, tRNA or degraded mRNA

In the FirstChoice RLM-RACE procedure, full-length mRNAs are selected by treating total or poly(A) RNA with FastAP Thermosensitive Alkaline Phosphatase to remove the 5'-phosphate from all molecules which contain free 5'-phosphates (ribosomal RNA, fragmented mRNA, tRNA, and contaminating genomic DNA). Full-length mRNAs are unaffected. The RNA is then treated with tobacco acid pyrophosphatase (TAP) to remove the cap structure from the full-length mRNA leaving a 5'-monophosphate. A synthetic RNA adapter is ligated to the RNA population—only molecules containing a 5'-phosphate, the uncapped, full-length mRNAs, will accept the adapter. Random-primed, reverse transcription reactions and nested PCR are then performed to amplify the 5'-end of your specific transcript.

From RNA to PCR in less than a day

Each step of the FirstChoice RLM-RACE procedure has been optimized, so you can complete all the enzymatic reactions and even start PCR in no more than 5 hours. The FirstChoice RLM-RACE Kit contains reagents and enzymes to produce 6 RLM-RACE-ready cDNA preparations, primers and nested RACE adapter primers for 100 PCR reactions. Kit includes reverse transcription reagents and 3' RACE Adapter and Primers for 3' RACE. Each kit contains control RNA and primers to test the kit's performance, protocol described in User Guide utilizing Platinum™ II Hot-Start PCR Master Mix.

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Shipping Condition: Dry ice