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

Product Code. 10277632
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Product Code. 10277632 Supplier Invitrogen™ Supplier No. 18373019

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Description

Includes

3' RACE System components are listed in Table 1.

Converts up to 1μg of total RNA preparation or 100ng of control RNA into cDNA in one reaction

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. 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.

  • Can be used with 5' RACE System to clone full-length sequences
  • Uses advanced capabilities of SuperScriptII RT for greater first-strand cDNA yields
  • Includes Control RNA and primers to monitor performance of system
  • Provides detailed protocols in manual

Cloning, PCR and Real-Time PCR, Reverse Transcription, cDNA Libraries and Library Construction

TRUSTED_SUSTAINABILITY

Specifications

Product Type cDNA library
Content And Storage 3´ RACE System components are listed in Table 1. Store the system at -20°C. Guaranteed stable for 6 months when properly stored.
For Use With (Application) cDNA Libraries & Library Construction
Quantity 20 Reactions

Frequently Asked Questions (FAQs)

Can I purchase the Adapter Primer (AP), Universal Amplification Primer (UAP), and Abridged Universal Amplification Primer (AUAP) from the 3‘ RACE System as stand-alone items?

The Adapter Primer (AP), Universal Amplification Primer (UAP), and Abridged Universal Amplification Primer (AUAP) have been discontinued as stand-alone items. Their sequences can be found on Page 4 of the manual.
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.

How does the 3' RACE system work?

3' RACE takes advantage of the natural poly(A) tail found in mRNA as a generic priming site for PCR. In this procedure, mRNAs are converted into cDNA using reverse transcriptase (RT) and an oligo-dT adapter primer. Specific cDNA is then amplified by PCR using a gene-specific primer (GSP) that anneals to a region of known exon sequences and an adapter primer that targets the poly(A) tail region. This permits the capture of unknown 3'-mRNA sequences that lie between the exon and the poly(A) tail.

What are some ways for me to optimize my RACE reaction?

Here are our suggestions to optimize your RACE reaction:

-Use high-quality, intact RNA
-Use an RNA sample in which your gene is expressed at high levels
-Do not exceed the recommended amount of input material in each step
-Optimize annealing temperatures for PCR
-Include recommended controls

Why does my RACE reaction show more than one band?

Alternative splicing, an alternative polyadenylation site, and alternative start sites can yield legitimate multiple bands. Sequencing will help to resolve any uncertainty.

How do I know if my bands from RACE are real?

If the reaction gives a specific product and the control reaction does not, your band is probably real. The only way to be sure is to sequence the product.

How do I design my gene-specific primers for my GeneRacer experiment?

If you are performing either 5' or 3' RACE, you will need one gene-specific primer and if you are performing both 5' and 3' RACE, you would need two gene-specific primers. The primers should follow the rules stated below:

-50-70% GC content to obtain a high annealing temp (>72 degrees C)
-23-28 nucleotides in length to increase specificity of binding
-Low-GC content at 3' ends to minimize extension by DNA polymerase at non-target sites (no more than two G or C residues in the last five bases)
-No self-complementary sequences within the primer or no sequence complementary to the primers supplied in the kit, especially at the 3' end
-Annealing temperature greater than 72 degrees C—using primers with a high annealing temperature will help to improve specificity of your PCR

How can I check the integrity of my starting RNA sample for RACE?

To check the RNA for integrity, analyze 500 ng of your RNA by agarose/ethidium bromide gel electrophoresis. You may use a regular 1% agarose gel or a denaturing agarose gel. For total RNA you should see the 28S and 18S rRNA bands. mRNA will appear as a smear from 0.5 to 12 kb. The 28S band should be twice the intensity of the 18S band. If you do not load enough RNA, the 28S band may appear to be diffuse. If you are using a denaturing gel, the rRNA bands should appear very clear and sharp. The 28S band should run at 4.5 kb and the 18S band should run at 1.9 kb.

What is RACE and what does it stand for?

RACE stands for Rapid Amplification of cDNA Ends. It is a method used to discover the 5' and/or 3' end of full-length transcripts. If partial sequence is known for a transcript of interest, RACE can help to elucidate the full ORF, 5' UTR, and 3' UTR sequences.

For Research Use Only. Not for use in diagnostic procedures.

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