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FirstChoice™ RLM-RACE Kit with Manual (Invitrogen™)

The FirstChoice® RLM-RACE Kit is designed to amplify cDNA only from full-length, capped mRNA, usually producing a single band after PCR. This kit is a major improvement over the basic rapid amplification of cDNA ends (RACE) protocol. The RLM-RACE procedure selects only full-length mRNA—no rRNA, tRNA or degraded RNA—and facilitates the cloning of sequences from the 5' ends of messages.

Features of the FirstChoice® RLM-RACE Kit:

• From RNA to PCR product in less than a day
• Yields single, specific product from rare transcripts
• Selects 5' and/or 3' ends of true messages
• Efficient—all enzymatic reactions are optimized to ensure detection of even the rarest mRNA

RACE
Rapid amplification of cDNA ends (5'-RACE) is a polymerase chain reaction-based technique developed to facilitate the cloning of sequence from the 5'-ends of messages. The FirstChoice® RLM-RACE Kit is a major improvement to the basic RACE protocol.

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 calf intestinal phosphatase (CIP) 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 5 RLM-RACE-ready cDNA preparations, in addition to primers and nested RACE adapter primers for 100 PCR reactions. Reverse transcription reagents are also included. Each kit contains control RNA and primers to test the kit's performance along with a detailed Instruction Manual. SuperTaq™ Thermostable Taq DNA Polymerase is available separately. For optimal amplification of fragments ≥1 kb, use SuperTaq-Plus. Also included are a 3' RACE Adapter and Primers for 3' RACE.

FirstChoice™ RLM-RACE Kit (Invitrogen™)

The FirstChoice® RLM-RACE Kit is designed to amplify cDNA only from full-length, capped mRNA, usually producing a single band after PCR. This kit is a major improvement over the basic rapid amplification of cDNA ends (RACE) protocol. The RLM-RACE procedure selects only full-length mRNA—no rRNA, tRNA or degraded RNA—and facilitates the cloning of sequences from the 5' ends of messages.

Features of the FirstChoice® RLM-RACE Kit:

• From RNA to PCR product in less than a day
• Yields single, specific product from rare transcripts
• Selects 5' and/or 3' ends of true messages
• Efficient—all enzymatic reactions are optimized to ensure detection of even the rarest mRNA

RACE
Rapid amplification of cDNA ends (5'-RACE) is a polymerase chain reaction-based technique developed to facilitate the cloning of sequence from the 5'-ends of messages. The FirstChoice® RLM-RACE Kit is a major improvement to the basic RACE protocol.

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 calf intestinal phosphatase (CIP) 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 5 RLM-RACE-ready cDNA preparations, in addition to primers and nested RACE adapter primers for 100 PCR reactions. Reverse transcription reagents are also included. Each kit contains control RNA and primers to test the kit's performance along with a detailed Instruction Manual. SuperTaq™ Thermostable Taq DNA Polymerase is available separately. For optimal amplification of fragments ≥1 kb, use SuperTaq-Plus. Also included are a 3' RACE Adapter and Primers for 3' RACE.

3' RACE System for Rapid Amplification of cDNA Ends (Invitrogen™)

3´ RACE System is suitable for rapid amplification of cDNA ends (RACE) (1-3) and anchored PCR between a defined point within mRNA and the 3´ poly(A) end (Figure 1). The system is useful for the amplification of rare messages for which little sequence information is available, and for capturing the 3´ end information of mRNA. The 3´ RACE System:

• Can be used with the 5´ RACE System to clone full-length sequences
• Uses the advanced capabilities of SuperScript® II RT for greater first-strand cDNA yields
• Includes Control RNA and primers to monitor the performance of the system
• Provides detailed protocols in the manual
• Converts up to 1 µg of total RNA preparation or 100 ng of control RNA into cDNA in one reaction

GeneRacer™ Kit with SuperScript™ III RT and Zero Blunt™ TOPO™ PCR Cloning Kit for Sequencing (Invitrogen™)

GeneRacer® is an advanced RACE (rapid amplification of cDNA ends) technique that improves the efficiency of amplifying full-length 5´ and 3´ cDNA ends. With the GeneRacer® Kit you can:

• Generate cDNA from transcripts at least 10 kb in length
• Obtain the full-length 5´ end of rare transcripts at fewer than 30 copies per cell
• Clone the full-length 5´ and 3´ ends to construct complete cDNA sequence

The GeneRacer® Kit is available with SuperScript® III Reverse Transcriptase (RT) for improved amplification of the full-length 5´ end from long and complex mRNA. The RNase H portion of SuperScript® III RT has been mutated to avoid cleaving mRNA during cDNA synthesis. This increases the size and yield of cDNA. SuperScript® III RT is more thermostable than wild-type RTs. This enables reverse transcription at higher temperatures, relaxing secondary structure of complex templates, and allowing cDNA synthesis to go to completion.

How GeneRacer® Works

The GeneRacer® Kit ensures that only transcripts containing full-length cDNA ends are amplified (1,2). Figure 1 outlines how the GeneRacer® Kit works. The advanced protocol starts at the RNA level by specifically targeting only 5´ capped mRNA. In subsequent steps the cap is removed and replaced with the GeneRacer® RNA Oligo. During reverse transcription, the GeneRacer® RNA Oligo sequence is incorporated into the cDNA. Only cDNA that is completely reverse transcribed will contain this known sequence. 5´ RACE PCR is then performed using the GeneRacer® 5´ Primer specific to the GeneRacer® RNA Oligo sequence and a gene-specific primer. The result is amplified DNA that contains the full-length 5´ cDNA sequence.

Sensitivity and Length

To demonstrate the ability of the GeneRacer® Kit to capture the full-length 5fi cDNA end, the 5fi ends of genes with known transcriptional start sites were amplified. Starting with total RNA and following the GeneRacer® protocol, both long transcripts (10 kb) and rare messages present at 0.01%, or 30 copies per cell were amplified (Figure 2).

GeneRacer™ Kit with AMV RT and TOPO TA Cloning™ Kit for Sequencing (Invitrogen™)

GeneRacer® is an advanced RACE (rapid amplification of cDNA ends) technique that improves the efficiency of amplifying full-length 5´ and 3´ cDNA ends. With the GeneRacer® Kit you can:

• Generate cDNA from transcripts up to 10 kb in length
• Obtain the full-length 5´ end of rare transcripts at fewer than 30 copies per cell
• Clone the full-length 5´ and 3´ ends to construct complete cDNA sequence

The GeneRacer® Kit is available with SuperScript® III Reverse Transcriptase (RT) for improved amplification of the full-length 5´ end from long and complex mRNA. The RNase H portion of SuperScript® III RT has been mutated to avoid cleaving mRNA during cDNA synthesis. This increases the size and yield of cDNA. SuperScript® III RT is more thermostable than wild-type RTs. This enables reverse transcription at higher temperatures, relaxing secondary structure of complex templates, and allowing cDNA synthesis to go to completion.

How GeneRacer® Works

The GeneRacer® Kit ensures that only transcripts containing full-length cDNA ends are amplified (1,2). Figure 1 outlines how the GeneRacer® Kit works. The advanced protocol starts at the RNA level by specifically targeting only 5´ capped mRNA. In subsequent steps the cap is removed and replaced with the GeneRacer® RNA Oligo. During reverse transcription, the GeneRacer® RNA Oligo sequence is incorporated into the cDNA. Only cDNA that is completely reverse transcribed will contain this known sequence. 5´ RACE PCR is then performed using the GeneRacer® 5´ Primer specific to the GeneRacer® RNA Oligo sequence and a gene-specific primer. The result is amplified DNA that contains the full-length 5´ cDNA sequence.

Sensitivity and Length

To demonstrate the ability of the GeneRacer® Kit to capture the full-length 5fi cDNA end, the 5fi ends of genes with known transcriptional start sites were amplified. Starting with total RNA and following the GeneRacer® protocol, both long transcripts (10 kb) and rare messages present at 0.01%, or 30 copies per cell were amplified (Figure 2).

GeneRacer™ Kit with SuperScript™ III RT and TOPO TA Cloning™ Kit for Sequencing (Invitrogen™)

The GeneRacer™ Kit provides a method to obtain full-length 5' and 3' ends of cDNA using known cDNA sequence from expressed sequence tags (ESTs), subtracted cDNA, differential display, or library screening. The kit ensures the amplification of only full-length transcripts via elimination of truncated messages from the amplification process. RACE PCR products can be quickly and easily cloned using either the Zero Blunt® TOPO® PCR Cloning Kit for Sequencing (blunt-end PCR products) or the TOPO TA Cloning® for Sequencing Kit (PCR products with 3' A-overhangs). Using the protocols provided, the cDNA ends of rare (30 copies/cell) and long (9 kb) transcripts can be amplified and sequenced starting from 1 µg of total RNA. With the GeneRacer® Kit you can:

• Generate cDNA from transcripts at least 10 kb in length
• Obtain the full-length 5´ end of rare transcripts at fewer than 30 copies per cell
• Clone the full-length 5´ and 3´ ends to construct complete cDNA sequenceSuperScript® III RT
The GeneRacer® Kit is available with SuperScript® III Reverse Transcriptase (RT) for improved amplification of the full-length 5´ end from long and complex mRNA. The RNase H portion of SuperScript® III RT has been mutated to avoid cleaving mRNA during cDNA synthesis. This increases the size and yield of cDNA. SuperScript® III RT is more thermostable than wild-type RTs. This enables reverse transcription at higher temperatures, relaxing secondary structure of complex templates, and allowing cDNA synthesis to go to completion.How the GeneRacer® Kit works
The GeneRacer® Kit ensures that only transcripts containing full-length cDNA ends are amplified (see figure). The advanced protocol starts at the RNA level by specifically targeting only 5´ capped mRNA. In subsequent steps, the cap is removed and replaced with the GeneRacer® RNA Oligo. During reverse transcription, the GeneRacer® RNA Oligo sequence is incorporated into the cDNA. Only cDNA that is completely reverse transcribed will contain this known sequence. 5´ RACE PCR is then performed using the GeneRacer® 5´ Primer specific to the GeneRacer® RNA Oligo sequence and a gene-specific primer. The result is amplified DNA that contains the full-length 5´ cDNA sequence.Sensitivity and length
To demonstrate the ability of the GeneRacer® Kit to capture the full-length 5´ cDNA end, the 5´ ends of genes with known transcriptional start sites were amplified. Starting with total RNA and following the GeneRacer® protocol, both long transcripts (10 kb) and rare messages present at 0.01%, or 30 copies per cell, were amplified (see figure).

5' RACE System for Rapid Amplification of cDNA Ends, version 2.0 (Invitrogen™)

The 5´ RACE System for Rapid Amplification of cDNA Ends, Version 2.0, is suitable for rapid amplification of cDNA ends (RACE) between a defined point in the mRNA and the 5´ end. The 5' RACE System provides a set of prequalified reagents intended for synthesis of first-strand cDNA, purification of first-strand products, homopolymeric tailing, and preparation of target cDNA for subsequent amplification by PCR. Control RNA, DNA, and primers are provided for monitoring system performance. The system is useful for the amplification of rare messages for which little sequence information may be available, and for capturing the 5´ end information of mRNA. The 5´ RACE system:

• Uses the advanced capabilities of SuperScript® II RNase H- RT for greater first-strand cDNA yields and increased cDNA lengths
• Prepares specific cDNA from up to 1 µg of total or poly(A)+ RNA in one reaction
• Offers improved first-strand cDNA purification procedure that yields cDNA suitable for tailing
• Uses recombinant TdT with optimized reaction conditions in a PCR-compatible buffer to provide an efficient primer annealing site for PCR