FirstChoice™ RLM-RACE Kit
We have updated the composition of the kit by changing Calf intestinal phosphatase (CIP) to FastAP thermosensitive alkaline phosphatase. Also, we have updated the protocol by removing phenol/chloroform extraction and ethanol precipitation. If desired, the old protocol can be followed with the new component.
FirstChoice™ RLM-RACE Kit
Invitrogen™

FirstChoice™ RLM-RACE Kit

Designed to amplify cDNA only from full-length, capped mRNA, usually producing a single band after PCR
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Catalog NumberIncludes
AM1700Kit, 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, Nuclease-free Water
AM1700MKit with manual, 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, Nuclease-free Water
Catalog number AM1700
Price (USD)
968.00
Each
In stock
Add to cart
Includes:
Kit, 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, Nuclease-free Water
Recurring order eligible. Learn more »
Price (USD)
968.00
Each
Add to cart
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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.

For Research Use Only. Not for use in diagnostic procedures.
Specifications
Final Product TypecDNA
For Use With (Application)cDNA Libraries & Library Construction
IncludesKit, 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, Nuclease-free Water
Optimal Reaction Temperature42°C
Product LineAmbion™, FirstChoice™
Product TypeRLM-RACE Kit
Quantity1 Kit
Reverse TranscriptaseM-MLV
Shipping ConditionDry Ice
FormatKit
Unit SizeEach
Contents & Storage

•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
•Ammonium Acetate Stop Solution
•Components should be stored at -20°C. Nuclease-free Water may be stored at any temperature.

Have questions about this product? Ask our AI assisted search.
How can I check the integrity of my starting RNA sample for RACE?
How can I improve my RLM-RACE reactions when using FirstChoice RLM-RACE Kit (Cat. No. AM1700, AM1700M)?
What is RACE and what does it stand for?
How do I know if my bands are real when using FirstChoice RLM-RACE Kit (Cat. No. AM1700, AM1700M)?
Why would I get no band at all when using FirstChoice RLM-RACE Kit (Cat. No. AM1700, AM1700M)?
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Frequently asked questions (FAQs)

How long can I store the cDNA from my reverse transcription step?

You can store your cDNA at 2-6 degrees C for up to 24 hours. For long-term storage, store the cDNA at -15 to -25 degrees C and add EDTA to a final concentration of 1 mM to prevent degradation.

I'm getting PCR products from my 5' RACE, but they are not full length. What should I do?

The GeneRacer method is designed to ensure that only full-length messages are ligated to the GeneRacer RNA Oligo and PCR amplified after cDNA synthesis. It is highly recommended that you clone your RACE products and analyze at least 10-12 colonies to ensure that you isolate the longest message. Many genes do not have only one set of transcription start sites but rather multiple transcription start sites spanning sometimes just a few or other times a hundred or even more bases. Cloning of the RACE products and analyzing multiple colonies ensues that you detect the diversity of the heterogeneous transcription start sites of your gene. It is also possible that you might obtain PCR products that may not represent the full-length message for your gene. PCR products that do not represent full-length message may be obtained because:

-RNA degradation after the CIP reaction creates new truncated substrates with a 5' phosphate for ligation to the GeneRacer RNA Oligo. Be sure to take precautions to ensure that the RNA is not degraded.
-CIP dephosphorylation was incomplete. Increase the amount of CIP in the reaction or decrease the amount of RNA.
-PCR yielded a PCR artifact and not true ligation product. Optimize your PCR using the suggestions described above.

I'm seeing RACE PCR artifacts in my GeneRacer experiment. What am I doing wrong?

RACE PCR artifacts or nonspecific PCR bands can result from one or more of the following:

-Nonspecific binding of GSPs to other cDNAs resulting in the amplification of unrelated products as well as desired products.
-Nonspecific binding of GeneRacer primers to cDNA resulting in PCR products with GeneRacer primer sequence on both ends of the PCR product.
-RNA degradation.
-Contamination of PCR tubes or reagents.
Note: Artifacts usually result from less than optimal PCR conditions and can be identified in negative control PCR.

I'm getting unexpected bands after electrophoretic analysis of my amplified RT-PCR products. Can you please offer some suggestions?

Please see the following causes and suggestions:
Contamination by genomic DNA or an unexpected splice variant - Pretreat RNA with DNase I, amplification grade (Cat. No 18068015).
Design primers that anneal to sequences in exons on both sides of an intron or at the exon/exon boundary of the mRNA to differentiate between amplified cDNA and potential contaminating genomic DNA.
To test if products were derived from DNA, perform a minus RT control.
Nonspecific annealing of primers - Vary the PCR annealing conditions.
Use a hot-start PCR polymerase.
Optimize magnesium concentration for each template and primer combination.
Primers formed dimers - Design primers without complementary sequences at the 3' ends.

I'm getting no bands after electrophoretic analysis of my amplified RT-PCR products. Can you please offer some tips?

Please see the following causes and suggestions:

Procedural error in first-strand cDNA synthesis - Use high-quality RNA as a control to verify the efficiency of the first-strand reaction.
RNase contamination - Add control RNA to sample to determine if RNase is present in the first-strand reaction. Use an RNase inhibitor in the first-strand reaction.
Polysaccharide co-precipitation of RNA - Precipitate RNA with lithium chloride to remove polysaccharides, as described in Sambrook et al.
Target mRNA contains strong transcriptional pauses - Use random hexamers instead of oligo(dT) in the first-strand reaction, increase the temperature, and use PCR primers closer to the 3' terminus of the target cDNA.
Too little first-strand product was used in PCR - Use up to 10% of first-strand reaction per 50 mL PCR.
Gene-specific primer was used for first-strand synthesis - Try another set of GSP or switch to oligo(dT). Make sure the GSP is the antisense of the sequence.
Inhibitors of RT present - Remove inhibitors by ethanol precipitation of mRNA preparation before the first-strand reaction. Include a 70% (v/v) ethanol wash of the mRNA pellet. Note: inhibitors of RT include SDS, EDTA, guanidinium salts, formamide, sodium pyrophosphate, and spermidine.
RNA has been damaged or degraded - Ensure that high-quality, intact RNA is being used.
Annealing temperature is too high - Decrease temperature as necessary and/or use touchdown PCR.

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