RACE Kit

For life science research only.
Not for use in diagnostic procedures.
5/3 RACE Kit,

2nd Generation
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Content version: April 2014
For the rapid amplification of either 5 or 3 cDNA ends
Formulation containing Transcriptor Reverse Transcriptase and recombinant
Terminal Transferase
Cat. No. 03 353 621 001

Store the kit at 15 to 25C
www.roche-applied-science.com
Kit for 10 reactions
Table of Contents
1.
What this Product Does ........................................................................................................ 3

Number of Tests
Kit Contents
Storage and Stability
Additional Equipment and Reagents Required
Application
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2.
How To Use this Product ...................................................................................................... 5
2.1
2.2
Before You Begin

Handling Instructions
Sequence Information
Primer Design for 5 RACE
5 RACE Control Reaction
Primer Design for 3 RACE
3 RACE Control Reaction
Experimental Overview
3.
Experimental Protocol .......................................................................................................... 9
3.1
Experimental Protocol for 5 RACE

First-Strand cDNA Synthesis
Quantification of PCR Products
Poly(A) Tailing of First-Strand cDNA
PCR Amplification of dA-Tailed cDNA
PCR Control Reaction
Control of First-Strand
Control PCR Amplification of the dA-Tailed First-Strand cDNA
PCR Amplification of cDNA
3.2
4.
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9
11
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14
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16
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Troubleshooting.................................................................................................................... 19
cDNA Synthesis
3 RACE
5.
5
5
5
5
6
7
7
8
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Additional Information on this Product........................................................................... 23

How this Product Works
5 RACE Overview
Test Principle
3 RACE Overview
Test Principle
References
Quality Control
23
24
25
26
26
27
27
6.
Supplementary Information............................................................................................... 28
6.1
Conventions
Text Conventions
Symbols
Changes to Previous Version
Ordering Information
License Limitations
Trademarks
Regulatory Disclaimer
6.2
6.3
6.4
6.5
6.6
5/3 RACE Kit, 2nd Generation
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1. What this Product Does
1.
What this Product Does
Number of Tests
Kit Contents
The kit is designed for 10 reactions for the rapid amplification of either 5 or 3
cDNA ends.
Vial/Cap Label
Contents / Function
1
purple
cDNA Synthesis
Buffer (5x conc.)
100 l
250 mM Tris-HCl, 40 mM MgCl2, 150
mM KCl, 5 mM dithiothreitol, pH 8.5
(+20C)
2
purple
Transcriptor Reverse 10 l
Transcriptase
25 U/l, in 200 mM potassium phosphate, 2 mM dithiothreitol, 0.2% (v/v)
Triton X-100, 50% glycerol (v/v), pH 7.2
3
green
Deoxynucleotide
Mixture
50 l
mixture of dATP, dCTP, dGTP, dTTP
10 mM each, in Tris-HCl, pH 7.5 (+20C)
4
green
dATP
30 l
2 mM, in Tris-HCl, pH 7.5 (+20C)
5
green
Reaction Buffer
(10x conc.)
1,000 l
100 mM Tris-HCl, 15 mM MgCl2,
500 mM KCl, pH 8.3 (+20C)
6
green
Terminal
Transferase,
recombinant
10 l
80 U/l in 60 mM potassium phosphate
(pH 7.2 at +4C), 150 mM KCl, 1 mM 2mercaptoethanol, 0.5% Triton X-100,
50% glycerol.
7
blue
Control neo-RNA
20 l
1 ng/l, in double-distilled water
8
red
Oligo dT-Anchor
Primer
40 l
37.5 M, in double-distilled water
9
red
PCR Anchor Primer
40 l
10
blue
Control Primer
neo1/rev
40 l
11
blue
Control Primer
neo2/rev
40 l,
12
blue
Control Primer
neo3/for
40 l
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1. What this Product Does

Storage and
Stability
Store the kit at 15 to 25C until the expiration date printed on the label.
Additional
Equipment and
Reagents
Required
For PCR amplification of poly(A)-tailed first-strand cDNA, the following

reagents are required:
Blends of thermostable DNA polymerases with improved fidelity and performance, Expand High Fidelity PCR System* or Expand Long Template PCR
System*
Whether Expand High Fidelity or Expand Long Template PCR System
should be used depends on the expected size of the PCR product and the
amount of template cDNA present in the reaction. The final concentration
of dNTP and MgCl2 should be adjusted according to the protocols given in
the individual Instructions for Use. If you want to use the Expand Long
Template PCR System and you need to establish a new assay, it is advisable to test all three possible amplification systems to find the optimum
reaction conditions.
High Pure PCR Product Purification Kit*
Thermal block cycler
Protector RNase Inhibitor* (optional)
Ethanol (pro analysis)
Mineral oil
Application
The kit is used for analysis of mRNA structure and expression using RACE
(rapid amplification of cDNA ends).
Generation of full-length cDNAs
Isolation and characterization of 5or 3 ends from low-copy RNA messages
Amplification and further cloning of rare mRNAs
Analysis in conjunction with exon-trapping methods
Products of the RACE reaction can be directly sequenced without any further
cloning step
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2. How To Use this Product
2.
How To Use this Product
2.1
Before You Begin
Handling
Instructions
To prevent degradation of RNA, autoclave all vessels and pipettes used for
cDNA synthesis.
Wear gloves when performing the experiment.
Use Protector RNase Inhibitor* to prevent degradation of RNA during firststrand cDNA synthesis.
The kit enables the transcription of poly(A)+ RNA into first-strand cDNA. The
Oligo(dT)-Anchor Primer effectively selects for polyadenylated RNA by initiating cDNA synthesis from the 5 start site of the poly(A) tail.
Allow all reagents required for reverse transcription to thaw completely.
Mix well and centrifuge briefly to collect the solutions at the bottom of the
vials.
Keep the reagents on ice while performing the assay; store them at 15 to
25C after the experiment.
Sequence
Information
Primer Design
for 5 RACE
Vial/Cap
Sequence
Vial 8 (red):
Oligo d(T)-Anchor Primer
5-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV-3
Mlu I site Cla I site Sal I site, V= A, C, or G
Vial 9 (red):
PCR Anchor Primer
5-GACCACGCGTATCGATGTCGAC-3
Vial 10 (blue):
Control Primer neo1/rev
5-CAGGCATCGCCATGGGTCAC-3
Nco I site
Vial 11 (blue):
Control Primer neo2/rev
5-GCTGCCTCGTCCTGCAGTTC-3
Pst I site
Vial 12 (blue):
Control Primer neo3/for
5-GATTGCACGCAGGTTCTCCG-3
At least two antisense gene-specific primers are needed.

Gene-specific primer SP1 is required to transcribe the mRNA into first-strand
cDNA.
A second, nested primer SP2 located upstream of SP1 is used for the first
PCR amplification. For a second PCR round, use a further nested primer SP3.
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5 RACE Control
Reaction
Perform a control reaction when working with the 5/3 RACE Kit the first time.
The kit contains a control system, including Control neo-RNA and three gene
specific neo-primers. The Control neo-RNA is an in vitro transcribed RNA from
the neomycin resistance gene that contains a 3 poly(A)+ tail and is 1,000
bases in size.
To your sample RNA, add the Control neo-RNA to test whether the cDNA
synthesis, the purification, the dA-tailing and the following PCR amplification
are working. Handle your RNA preparation and our nuclease-free Control
neo-RNA together in one tube to check for presence of contaminating
nucleases. Determine the sensitivity of the 5/3 RACE Kit by using dilutions of
the control RNA.
Transcribe the Control neo-RNA into a 655 bp first-strand cDNA using the
Control Primer neo 1/rev. To check the efficiency of the cDNA synthesis, the
cDNA is amplified using the Control Primer neo 2/rev and Control Primer
neo 3/for, obtaining a 157 bp PCR product. To check the purification efficiency, perform this control PCR before and after the purification step.
Efficiency of the dA-tailing reaction of the purified cDNA is checked by a
control PCR using Oligo(dT)-Anchor Primer and neo2/rev Primer. Specific
amplification of the dA-tailed control cDNA results in a prominent 293 bp
fragment. Alternatively, this PCR assay may be used to optimize PCR parameters.
Reaction
Primer
Resulting PCR Product
cDNA synthesis
neo1/rev
655 bp
Control of cDNA synthesis: neo3/for

neo2/rev
157 bp
Amplification of dA-tailed
cDNA
293 bp
neo2/rev
Oligo (dT)-Anchor
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Primer Design
for 3 RACE
A gene-specific forward primer SP5 is required. Efficient and specific PCR

amplification is highly dependent on effective primer design. Primer design is
aimed at obtaining a balance between two goals: specificity and efficiency of
amplification. Therefore, the potential for secondary structure and dimer formation should be minimized. The desirable primer length is 20 to 25 bp, with a
GC content of 50 to 60%. For further information, see Saiki (4) and Frohmann
(5).
3 RACE Control
Reaction
First-strand cDNA synthesis is initiated at the poly(A)+ tail of the neo-RNA

using the Oligo(dT)-Anchor Primer, obtaining a 1,040 bp cDNA. The firststrand cDNA can be directly amplified by PCR with the PCR Anchor Primer
and the neo3/for Primer, resulting in a 1,026 bp PCR product.
Reaction
Primer
Resulting PCR Product
cDNA synthesis
Oligo(dT)-Anchor Primer 1,040 bp
Amplification of cDNA neo3/for

PCR Anchor Primer
1,026 bp
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2.2
Experimental Overview
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3. Experimental Protocol
3.
Experimental Protocol
3.1
First-Strand
cDNA Synthesis
Component
Volume
cDNA Synthesis Buffer (Vial 1)
4 l
Deoxynucleotide Mixture (Vial 3)
2 l
cDNA synthesis primer SP1 (12.5 M)
1 l
poly(A) RNA or total RNA 0.2 2 g
x l
For control reaction:

Control Primer neo1/rev (Vial 10)
Control neo-RNA (Vial 7)
1 l
1 l
Transcriptor Reverse Transcriptase (vial 2)
1 l
Double-distilled water
x l
Total Volume
20 l
Mix the reaction mixture and spin down briefly.

Incubate for 60 min at +55C.
Incubate another 5 min at +85C.
Briefly spin down the mixture.
Remove 1 l for later PCR control reaction.
Do not interrupt the protocol or store the cDNA because singlestranded cDNA is much more fragile than dsDNA. Directly proceed
with cDNA purification.
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Starting from total RNA or poly(A)+ RNA, the kit enables the transcription of
specific mRNA sequences into first-strand cDNA. In general, total RNA is used
as starting sample material, but the usage of poly(A)+ RNA may be advantageous for decreasing background or to enrich very rare messages. Transcriptor Reverse Transcriptase is provided in the kit, because of its increased heat
stability compared to other reverse transcriptases, as well as its ability to
reverse transcribe mRNA up to 14 kb in length. Therefore, the incubation temperature can be raised to +55C to encourage reverse transcription to proceed
through regions of difficult secondary RNA structure or high GC content.
Applying such stringent reaction conditions will result in highly efficient cDNA
synthesis.
Do not interrupt the protocol or store the cDNA because single-stranded
cDNA is much more fragile than dsDNA. Directly proceed with cDNA
purification.
Pipet the following into a sterile microcentrifuge tube on ice:
For the following protocol, use the reagents from the High Pure PCR Product
Purification Kit.
It is important to use this special protocol and not the protocol from the
Instructions for Use of the High Pure PCR Product Purification Kit. Add 40
ml ethanol p. a. to the Wash Buffer of the pack size of 50 purifications of
the High Pure PCR Product Purification Kit. The Binding Buffer (green cap)
contains guanidine thiocyanate, which is an irritant. Wear gloves and follow laboratory safety conditions during handling.
Add 100 l Binding Buffer (green cap) to 20 l of the first-strand
cDNA reaction and mix well.
Protocol for the Purification of cDNA
Combine the High Pure Filter Tube and the Collection Tube and pipet
sample into the upper reservoir.
Centrifuge for 30 sec at 6,000 to 8,000 g in an Eppendorf centrifuge.
Remove the Filter Tube from the Collection Tube.

Discard the flow through liquid in the Collection Tube.
Reinsert the Filter Tube into the same Collection Tube.
Add 500 l Wash Buffer to the upper reservoir of the Filter Tube.
Make sure that the Filter Tube has no contact with the surface of the
Wash Buffer flow through.
Discard the flow through liquid in the Collection Tube.
Reinsert the Filter Tube into the same Collection Tube.
Add 200 l Wash Buffer to the upper reservoir of the Filter Tube.
Centrifuge at least 2 min at maximum speed (approximately 13,000

g) in an Eppendorf centrifuge.
This additional washing step with reduced buffer volume ensure
optimal purity and the complete removal of residual wash buffer
from the glass fiber fleece.

Discard the Collection Tube with the flow through.
Insert the Filter Tube into a sterile 1.5 ml microcentrifuge tube.

Add 50 l Elution Buffer (Vial 3) to the Filter.
The microcentrifuge tube contains the eluted cDNA.

Remove 1 l of the purified cDNA from the microcentrifuge tube for
later PCR control reaction.
Use the purified cDNA directly for poly(A) tailing by Terminal Transferase.
Do not interrupt the protocol or store the cDNA because singlestranded cDNA is much more fragile than dsDNA.
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Quantification of
PCR Products
The concentration and purity of PCR product can be determined by spectrophotometric measurement at 260 nm and 280 nm.
In rare cases, glass fibers from the filter column may co-elute together
with the cDNA; this can disturb subsequent UV absorbance measurements. Therefore, centrifuge the tube with the eluted cDNA for 1 to 2 min
at high speed and carefully pipet from the surface.
Poly(A) Tailing of The following protocol describes the addition of a homopolymeric A-tail to the
3 end of first-strand cDNA using recombinant Terminal Transferase and
First-Strand
dATP.
cDNA
Pipet the following into a sterile microcentrifuge tube on ice.
Volume
19 l
Reaction Buffer, 10x conc. (Vial 5)
2.5 l
2 mM dATP (Vial 4)
2.5 l
Poly(A) Tailing of First-Strand cDNA
Component
Purified cDNA sample

Chill on ice.
Add 1 l of Terminal Transferase rec. (80 U/l, Vial 6).
Mix and incubate at +37C for 20 min.
The incubation time may be increased up to 30 min.
Incubate at +70C for 10 min to heat inactivate the Terminal Transferase.
Briefly spin down the mixture and place the tube on ice.
PCR
Amplification of
dA-Tailed cDNA
In the following table, the amplification of dA-tailed cDNA using the Expand
High Fidelity PCR System* in a first and second optional (nested) PCR is
described. The tailed cDNA can be directly amplified by PCR without prior
purification or dilution.
If you want to use the Expand Long Template PCR System, follow the
detailed instructions given in the Instructions for Use. for a 50 l amplification reaction. Always use the primer and template concentrations given in
section Experimental Protocol.
The optimal reaction conditions depend on the template/primer pair and must
be determined individually. Use an annealing temperature close to the effective melting temperature of the primers.
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Component
Volume
dA-tailed cDNA
5 l
Oligo dT-Anchor Primer (Vial 8)
1 l
Specific primer SP2 (12.5 M)
1 l
Deoxynucleotide Mixture (Vial 3) 1 l

Expand High Fidelity
Enzyme Mix
0.75 l
Expand High Fidelity Buffer

(10) with 15 mM MgCl2 (supplied with the Expand High
Fidelity PCR System)
5 l
36.25 l
Total Volume
50 l

Overlay with 50 l mineral oil if necessary.
Place the reaction mix in a thermal block cycler and start PCR.
An example for temperature cycling conditions for the Perkin Elmer
Cetus DNA Thermal Cycler (Models 9600 and 2400) is given below
Temp.
Time
Cycle No.
Initial denaturation
94C
2 min
Denaturation
Annealing
Elongation
94C
55C1)
72C
15 sec
30 sec 10
40 sec
Denaturation
Annealing
Elongation
94C
55C1)
72C
15 sec
30 sec 25
40 sec2)
Final elongation
72C
7 min
1)
Annealing temperature depends on the melting temperature of the primer used.

Elongate each successive cycle by additional 20 sec (i.e., elongation time of cycle no.
11 is 40 sec; cycle no. 12, 60 sec; cycle no. 13, 80 sec; etc.).
2)
If
Then
you end up with enough PCR product,
store at +2 to +8C
you used a cDNA for rare messages, you need

a second PCR round to obtain a visible
PCR product;
go ahead with
Steps 5 to 10 and
add a second PCR
round (nested
PCR).
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Dilute 10 l of the amplification product from the first round to 1:20 in
double-distilled water.
Amplify 1 l of the diluted material using the PCR Anchor Primer and a
nested gene-specific primer 3 in a second PCR. If there is insufficient
sequence information to design a nested primer, it is useful to reamplify
gel-purified, size-selected PCR products using the PCR Anchor Primer
and the original SP2 primer.
Second PCR round (nested PCR), if necessary.
Pipet the following into a sterile microfuge tube on ice:
Volume
Diluted or undiluted PCR

product
1 l
PCR Anchor Primer (Vial 9)
1 l
1 l
Component

Expand High Fidelity Enzyme
Mix
0.75 l

(10) with 15mM MgCl2 (supplied with the Expand High
5 l
40.25 l
Total Volume
50 l
Mix well.
Place the sample in a thermal block cycler and start PCR.
An example for temperature and cycle conditions is mentioned
above. Depending on the melting temperature of the gene-specific
primer SP3, increase the annealing temperature up to +60 to +65C.
Use 20 l of both the first and second PCR product for analysis on a 1%,
ethidium-bromide stained agarose gel with a corresponding DNA
molecular weight marker.
In general, only one major band is generated, but occasionally, a
background smear or a few minor bands might be visible. To verify
that the correct fragment has been amplified, perform Southern blot
hybridization analysis. If specific hybridization is not observed, see
section Troubleshooting.
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PCR Control
Reaction
If you use the Control neo-RNA in the cDNA synthesis, perform the following
PCR control reactions.
Control of FirstStrand
Use 1 l of the cDNA synthesis reaction assay (both before and after purification) as template for the PCR reaction.
Component
Volume
cDNA (purified or not purified)
1 l
Control of First-Strand
Control Primer neo2/rev (Vial 11) 1 l

Control Primer neo3/for (Vial 12)
1 l
1 l
Expand High Fidelity Enzyme Mix 0.75 l

Expand High Fidelity Buffer (10) 5 l
with 15 mM MgCl2 (supplied with
the Expand High Fidelity PCR
System)
40.25 l
Total Volume
50 l
Mix and spin down briefly.

Overlay with mineral oil if necessary.
Use 20 l of both the first and second PCR product for analysis on a 1%
ethidium-bromide stained agarose gel with a corresponding molecular
weight standard.
If the cDNA synthesis and purification step have been successful, a
strong PCR product band of 157 bp should be visible. If this is not the
case, see section Troubleshooting.
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Control PCR
Amplification of
the dA-Tailed
First-Strand
cDNA
Amplify 1 l dA-tailed cDNA by using the PCR Oligo dT-Anchor Primer and
the Control Primer neo2/rev.
Component
Volume
1 l
1 l
Control PCR Amplification
dA-Tailed DNA
Oligo dT Anchor Primer (Vial 8)
Control Primer neo2/rev (Vial 11) 1 l

1 l
Expand High Fidelity Enzyme Mix 0.75 l

Expand High Fidelity Buffer (10) 5 l
with 15 mM MgCl2 (supplied with
the Expand High Fidelity PCR
System)
40.25 l
Total Volume
50 l

Overlay with mineral oil if necessary.
Use 20 l of both the first and second PCR product for analysis on a 1%
ethidium bromide-stained agarose gel with a corresponding molecular
weight standard.
If the dA-tailing reaction has been successful, a strong band of 293
bp should be visible after amplification.
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3.2
First-Strand
cDNA Synthesis

Component
Volume
cDNA Synthesis Buffer (Vial 1)
4 l
2 l
Oligo dT-Anchor Primer (Vial 8)
1 l
poly(A)+
0.5 to 2 g
RNA or total RNA
[for control reaction: Control neo-RNA (Vial 7)]
[1 l ]
Transcriptor Reverse Transcriptase (Vial 2)
1 l
x l
Total Volume
20 l

Incubate additionally for 5 min at +85C.
PCR
Amplification of
cDNA
The cDNA can be directly amplified by PCR without prior purification. Use 1 l
of the cDNA reaction mix, the PCR Anchor Primer and a gene-specific primer
SP5 in the PCR reaction. The optimal reaction conditions depend on the template/primer pair and must be determined individually. Use an annealing temperature from +60 to +65C.
Whether Expand High Fidelity or Expand Long Template PCR System
should be used depends on the expected size of the PCR product and the
amount of template cDNA present in the reaction. The final concentration
of dNTP and MgCl2 should be adjusted according to the protocols given in
the individual instructions for use. If you want to use the Expand Long
Template PCR System and you need to establish a new assay, it is advisable to test all three possible amplification systems to find the optimum
reaction conditions.
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Component
Volume
1 l
1 l
1 l
PCR Amplification of cDNA
cDNA product

Mix
0.75 l

5 l
40.25 l
Total Volume
50 l

Use 20 l of the PCR amplification product for analysis on a 1% ethidium bromide-stained agarose gel with a corresponding DNA molecular
weight marker.
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3.2
Experimental Protocol for 3 RACE, continued
PCR Control
Reaction
If you have used the Control neo-RNA in the cDNA synthesis reaction, use the
following PCR control reaction. Use 1 l of the cDNA synthesis reaction assay,
the provided neo3/for primer and the PCR Anchor Primer in the PCR reaction.
Component
Volume
cDNA product
1 l
1 l
Control Primer neo3/for (Vial 12) 1 l

Mix
0.75 l

5 l
40.25 l
Total Volume
50 l
Mix and spin down briefly..

Use 20 l of the PCR amplification product for analysis on a 1%, ethidium bromide-stained agarose gel with a corresponding DNA molecular
weight marker.
If the 3 RACE amplification has been successful, a strong band of
1,026 bp should be visible. If this is not the case, see section
Troubleshooting.
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4. Troubleshooting
4.
Troubleshooting
cDNA Synthesis
Low or no
product
Possible Cause
Recommendation
Reagents contaminated
Verify that your PCR system works well performing

a common PCR reaction using a DNA template
and primers to ensure the integrity of the reagents.
RNA is degraded or of poor Electrophorese the RNA of interest in a 1% formquality.

aldehyde minigel and examine integrity of the 18S
and 28S ribosomal bands. If the RNA is degraded
or of poor quality, isolate a new total RNA (6).
RNase contamination
Successful cDNA synthesis demands RNase-free

handling at all times. Wear gloves to avoid contamination of the kit components, use sterilized
pipettes and tubes. For more details, see (6, 7).
If necessary, use Protector RNase Inhibitor* during first-strand cDNA synthesis.
Reverse transcription is
Ensure that the RNA preparation is free of agents
inhibited by contamination. that inhibit reverse transcription, such as phenol,
lithium chloride and SDS (8).
RNA preparation is
contaminated with
genomic DNA.
Make sure that the RNA preparation is free of

contaminating genomic DNA. If necessary, perform a control experiment without the cDNA synthesis step; any obtained PCR products result
from amplification of genomic DNA.
Purification steps not

efficient
If you do not obtain a strong 157 bp PCR product

using the purified control cDNA as template,
make sure that the purification procedure is correct.
Reagents contaminated
Verify that your PCR system works well performing

a common PCR reaction using a DNA template
and primers to ensure the integrity of the reagents.
Concentration of the
Perform a Southern blot analysis of the PCR prodspecific product too low for uct using internal sequences as probe to identify
detection by ethidium
specific product bands.
bromide staining
Try a second PCR round using a nested primer
SP4 and the PCR Anchor Primer.
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4. Troubleshooting
Low or no
product
Possible Cause
Recommendation
Purification steps not

efficient
Use an agarose gel-purified PCR product as template in the second PCR for more specific amplification products.
Use the High Pure PCR Product Purification Kit
between the first and second PCR round for more
specific amplification products.
Nonspecific products are

being amplified.
Raise the annealing temperature gradually until

nonspecific products are no longer observed.
Improper cDNA
denaturation.
Check if the denaturation step prior to the tailing

reaction was performed properly according the
protocol: Incubate for 3 min at +94C. Chill on ice.
Insufficient incubation time The incubation period for the tailing reaction can
for poly(A) tailing.
be increased to 30 min.
A260 nm reading of nucleic

acid eluate
too high.
Improper storage of the

High Pure PCR Product
Purification Kit.
If the High Pure PCR Product Purification Kit is not

stored at +15 to +25C but at +2 to +8C, precipitation within the Binding Buffer may occur. Such
precipitates of buffer components may be carried
over to the final cDNA eluate and inhibit the subsequent Terminale Transferase reaction.
Incorrect cDNA purification protocol.
Follow the purification protocol in the Instructions

for Use of the 5/3 RACE, not the High Pure PCR
Product Purification Kit. It is crucial that centrifugation after the last washing step (prior to elution)
is performed at maximum speed of 13,000 g and
for a mininum of 2 min. The Filter Tube should be
completely dry before elution of bound cDNA.
Otherwise, residual ethanol inhibits the tailing
reaction. All other centriguation steps should be
performed at 6,000 to 8,000 g and here 30 sec
duration is sufficient.
Glass fibers, which might

co-elute with nucleic acid
and disturb absorbance
measurement.
1. Remove High Pure Filter Tube from tube containing eluted sample and spin sample for 1 min at
maximum speed.
2. Use an aliquot of the supernatant. Do not disturb the glass fibers at the bottom of the original
tube.
3. It may be useful to make a correction by subtracting the value measured at 320 nm from the
value measured at 260 nm.
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4. Troubleshooting
Possible Cause
Stretch of dTs Wrong annealing
in the 5
conditions
region of the
final PCR
product
Insufficient purification of
cDNA.
Recommendation
If the annealing conditions during the first PCR
amplification are not optimal (i.e., the anchor
primer is not completely annealed and might bind
within the 5poly(A) tail) and a proofreading polymerase (e.g., Expand High Fidelity PCR System or
Expand Long Template PCR System) is used, the 3
5exonuclease activity might remove a nonannealed 3 non-T anchor base. This would lead to
priming of PCR from within the poly(A) tail and the
introduction of long 5 T stretches into the PCR
product. Stringent annealing conditions are crucial
for success of the reaction.
Correct purification of the first-strand cDNA is
extremely important to remove any residual unincorporated nucleotides. Otherwise, in the 5 tailing
reaction, other nucleotides than A could be incorporated, which would lead to internal binding of
the Oligo-d(T) Anchor Primer to the 5 poly(A) tail.
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4. Troubleshooting
3 RACE
No PCR amplification
product using the
control system or no
specific PCR amplification product with
your own RNA/primer
system.
Possible Cause
Recommendation
Reagents
contaminated
Verify that your PCR system in general works

well by performing a common PCR reaction
using a DNA template and primers to ensure
the integrity of the reagents.
RNA degraded or of
poor quality
Electrophorese the RNA in a 1% formaldehyde

minigel and examine integrity of the 18S and
28S ribosomal bands. If the RNA is degraded or
of poor quality, isolate a new total RNA (6).
RNase contamination Successful cDNA synthesis demands RNasefree handling at all times.
Wear gloves to avoid contamination of the kit
components and use sterilized pipettes and
tubes. For more details, see (6, 7).
If necessary, use Protector RNase-Inhibitor*
during first-strand cDNA synthesis.
Only low yields of

specific products are
observed.
Reverse transcription
is inhibited by contamination.
Ensure that the RNA preparation is free of

agents that inhibit reverse transcription, such
as phenol, lithium chloride and SDS (8).
Contamination with
genomic DNA
Make sure that the RNA preparation is free of

contaminating genomic DNA.
If necessary, perform a control experiment
without the cDNA synthesis step; any obtained
PCR products result from amplification of
genomic DNA.
Concentration of the
specific product too
low for detection by
ethidium bromide
staining.
Perform a Southern blot analysis of the PCR

product using internal sequences as probe to
identify specific product bands.
Try a second PCR round using a nested primer
SP6 and the PCR Anchor Primer.
Non-specific
products are being
amplified.
Raise the annealing temperature gradually until

non-specific products are no longer observed.
22
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5. Additional Information on this Product
5.
Additional Information on this Product
How this Product Generating a full-length cDNA is of critical importance in studies on gene
structure and expression. An intact, full-length cDNA including the very 5 end
Works
is rarely recovered from cDNA libraries, despite time-consuming cDNA library
screening. Often, the 5 end of the cDNA strand is missing because of the
inability of the reverse transcriptases to read through an entire gene
sequence; this is a problem particularly in the case of extremely large gene
transcripts. Therefore, methods have been developed to amplify DNA
sequences from a messenger RNA (mRNA) template between a defined internal site and unknown sequences of either the 3 or the 5 end of the mRNA.
These methods are often referred to as RACE (rapid amplification of cDNA
ends), anchored PCR (1), or one-sided PCR (2), and were first described by
Frohmann et al. (3).
23
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5 RACE Overview
Overview / 5 RACE
mRNA
(A)n-3
SP 1
cDNA
(A)n-3
Purify cDNA with

High Pure PCR Product Purification Kit
3-(A)n AAAA
synthesis of first strand

cDNA with primer SP 1
degradation of the mRNA

template by the RNase H
activity of Transcriptor
Reverse Transcriptase
tailing of the purified cDNA

with dATP and TdT
Oligo(dT)-Anchor primer
( T )n T T T T V
(A)n AAAA
amplification of the tailed

cDNA by PCR using the
Oligo(dT)-Anchor primer and
a nested SP 2 primer
SP 2
PCR Anchor
primer
second PCR with the PCR Anchor
primer and a SP3 primer
SP 3
PCR product ready for:

agarose gel electrophoresis
analysis by hybridisation or cloning
V = A, C or G
5 RACE allows the amplification of unknown sequences at the 5 end of the mRNA.
24
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Test Principle
First-strand cDNA synthesis

First-strand cDNA is synthesized from total or poly(A)+ RNA using a
gene-specific primer SP1, Transcriptor Reverse Transcriptase, and the
Deoxynucleotide Mixture. Transcriptor Reverse Transcriptase is used
because of its greater heat stability compared to the other reverse transcriptases and its ability to reverse transcribe up to 14 kb long mRNA.
Therefore, the incubation temperature for first-strand cDNA synthsis
can be raised up to +55C to encourage reverse transcription to proceed through regions of difficult secondary RNA structure.
Purification
The first-strand cDNA is purified from unincorporated nucleotides and
primers using the High Pure PCR Product Purification Kit*.
Addition of homopolymeric A-tail

Terminal transferase is used to add a homopolymeric A-tail to the 3
end of the cDNA. Since vertebrate coding sequences and
5 untranslated RNA regions tend to be biased toward G/C residues,
the use of a poly(A)-tail decreases the likelihood of inappropriate truncation by the Oligo dT-Anchor Primer (3). Additionally, poly(A)-tail is
used because A/T binding is weaker than G/C binding; therefore, longer stretches of A residues are required before the Oligo dT-Anchor
Primer will bind to an internal site and truncate the amplification product.
First PCR amplification

Tailed cDNA is then amplified by PCR using a gene-specific primer SP2
and the Oligo dT-Anchor Primer. The Oligo(dT)-Anchor Primer is a mixture of oligonucleotides carrying a non-T nucleotide (i.e., A, C, or G) at
the 3 end following the dT-stretch. By this means the Oligo(dT)Anchor Primer is forced to bind to the (5) start site of the poly(A)-tail.
Thus, the actual length of the poly(A)-tail has no influence on priming.
Second PCR amplification

The obtained cDNA is further amplified by a second PCR using a
nested, gene-specific primer SP3 and the PCR Anchor Primer. As a
result, the obtained 5 RACE products can be cloned into an appropriate vector for subsequent characterization procedures, which may
include sequencing and restriction mapping.
25
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3 RACE Overview
Overview / 3 RACE
mRNA
AAAAAA
VTTTT
cDNA
VTTTT
SP 5
by PCR using a gene-specific
PCR Anchor
primer
cDNA synthesis
using the oligo(dT)Anchor primer
degradation of the
mRNA by the RNase H
activity of Transcriptor
Reverse Transcriptase
amplification of the cDNA
primer SP 5 and the PCR
Anchor primer
PCR product ready for:

agarose gel electrophoresis or
cloning procedures
V = A, C or G
3 RACE takes advantage of the natural poly(A)-tail of mRNAs as a priming site for PCR amplification.
A Control neo-RNA and three control primers (neo1/rev, neo2/rev, and
neo3/for) are included in the kit to verify performance of the first-strand

cDNA synthesis, tailing reaction, and following amplification.
Test Principle
First-strand cDNA synthesis is initiated at the poly(A)-tail of mRNA

using the Oligo(dT)-Anchor Primer.
After converting mRNA into cDNA, the following amplification is then

directly performed without a further purification step using the PCR
Anchor Primer and a user-designed gene-specific primer (SP5).
26
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References
1
2
3
4
5
6
7
8
9
10
11
12
13
Quality Control
Loh,Y. et al. (1989) Science 243, 217.

Ohara et al. (1989) Proc. Natl. Acad. Sci USA 86, 5673.
Frohmann, M. (1994) PCR Methods and Applications 4, 540558.
Saiki, R. (1990) PCR Protocols: A Guide to Methods and Applications,
pp. 1320.
Frohmann, M. (1990) PCR Protocols: A Guide to Methods and Applications, pp. 2838.
Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156.
Molecular Cloning, A Laboratory Manual (1989, Nolan, C, ed.) 2nd
edition, pp 7.37.8 and 10.2710.37.
Current Protocols in Molecular Biology (1987, Ausubel et al. eds.) Vol 1,
pp 3.8 and 4, Massachusetts General Hospital, Havard Medical School.
David, M. et al. (2003) Critical role of the transcriptional repressor
neuron-restrictive silencer factor in the specific control of connexinin
insulin-producing cell lines. J. Biol. Chem. 278, 53082-9.
Benachour, A. et al. (2005) The enterococcus faecalis SigV protein is an
extracytoplasmic function sigma factor contributing to survival following
heat, acid, and ethanol treatments. J. Bacteriol. 187, 1022-35.
Golovine, K. et al. (2003) Three different promoters control expression of
the aromatase cytochrome P450 gene (Cyp19) in mouse gonads and
brain. Biol Reprod 68, 978-84.
Mouchel, N. et al. (2003) Alternative 5' exons of the CFTR gene show
developmental regulation. Hum. Mol. Genet. 12, 759-69.
Nie, G.Y. et al. (2003) A novel serine protease of the mammalian HtrA
family is up-regulated in mouse uterus coinciding with placentation.
Mol. Hum. Reprod. 9, 279-90.
3 RACE: Transcription of the Control neo-RNA using the Oligo(dT) Anchor

Primer. Amplification of cDNA using the anchor and the neo3/for primers,
obtaining a 1,026 bp PCR product.
5 RACE: Transcription of the Control neo-RNA using neo1/rev primer.
Amplification of cDNA without prior tailing using the neo3/for and neo2/rev
primers, obtaining a 157 bp PCR product. Amplification of cDNA after tailing
using the PCR Anchor and neo2/rev primers, obtaining a 293 bp
PCR product.
27
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6. Supplementary Information
6.
Supplementary Information
6.1
Conventions
Text Conventions To make information consistent and memorable, the following text conventions are used in this document:
Symbols
Text Convention
Use
Numbered stages
labeled , , etc.
Stages in a process that usually occur in the order

listed.
Numbered instructions
labeled , , etc.
Steps in a procedure that must be performed in

the order listed.
Asterisk *
Denotes a product available from Roche Applied

Science.
In this document, the following symbols are used to highlight important information:
Symbol
6.2
Description
Information Note:
Additional information about the current topic or procedure.
Important Note:
Information critical to the success of the procedure or use of the product.
Changes to Previous Version

Editorial changes
28
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6.3
Ordering Information
Roche Applied Science offers a large selection of reagents and systems for life
science research. For a complete overview of related products and manuals,
please visit and bookmark our homepage www.roche-applied-science.com
and our Special Interest Sites including: http://www.roche-applied-science.com/PCR
Product
Pack Size
Cat. No.
Kits
High Pure PCR Product
1 kit (50 purifications)
11 732 668 001
Purification Kit
1 kit (250 purifications) 11 732 676 001
High Pure RNA Isolation Kit
1 kit (50 isolations)
11 828 665 001
High Pure RNA Tissue Kit
1 kit (50 isolations)
12 033 674 001
mRNA Isolation Kit
1 kit
11 741 985 001
mRNA Isolation Kit for Blood/
1 kit
11 934 333 001
Bone Marrow
Rapid DNA Ligation Kit
1 kit (40 reactions)
11 635 379 001
Single reagents
Protector RNase Inhibitor
10,000 U
03 335 402 001
2,000 U
03 335 399 001
Transcriptor Reverse
250 U
03 531 317 001
Transcriptase
500 U
03 531 295 001
2,000 U
03 531 287 001
Terminale Transferase,
8,000 U
03 333 566 001
recombinant
24,000 U
03 333 574 001
Expand High Fidelity PCR
100 U
11 732 641 001
System
2 250 U
11 732 650 001
10 250 U
11 759 078 001
Expand Long Template PCR
150 U
11 681 834 001
System
720 U
11 681 842 001
3,600 U
11 759 060 001
Proteinase K, recombinant,
1.25 ml
03 115 887 001
PCR Grade
5 ml
03 115 828 001
25 ml
03 115 844 001
Agarose MP
100 g
11 388 983 001
500 g
11 388 991 001
DNA Molecular Weight Marker 50 g (1 A260 unit)
11 336 045 001
VIII
11 449 460 001
IX
11 721 925 001
XIII
11 721 933 001
XIV
29
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PCR Nucleotide
Selection
Product
Deoxynucleoside Triphosphate
Set
PCR Nucleotide Mix
PCR Nucleotide MixPLUS
Pack Size
4 250 l
4 1,250 l
200 l
10 200 l
2 100 l
Cat. No.
11 969 064 001
03 622 614 001
11 581 295 001
11 814 362 001
11 888 412 001
6.4
License Limitations
For patent license limitations for individual products please refer to:
www.technical-support.roche.com.
6.5
Trademarks
EXPAND and HIGH PURE are trademarks of Roche.
All other product names or trademarks are the property of their respective
owners.
6.6
Regulatory Disclaimer
For life science research only. Not for use in diagnostic procedures.
30
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Contact and Support
If you have questions or experience problems with this or any Roche Applied
Science (RAS) product, please contact our Technical Support staff. Our
scientists are committed to providing rapid and effective help.
Please also contact us if you have suggestions for enhancing RAS product
performance or using our products in new or specialized ways. Such customer
information has repeatedly proven invaluable to the research community
worldwide.
To ask questions, solve problems, suggest enhancements or report new applications, please visit our Online Technical Support Site.
Visit the Roche Applied Science homepage, www.roche-appliedscience.com, to download or request copies of the following materials:
Instructions for Use
Material Safety Data Sheets
Certificates of Analysis
Technical Manuals
Lab FAQS: Protocols and references for life science research
0414.035395630013
To call, write, fax, or email us, visit the Roche Applied Science homepage and
select your home country to display country-specific contact information.
Roche Diagnostics GmbH

Sandhofer Strasse 116
68305 Mannheim
Germany

RACE Kit

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Not for use in diagnostic procedures.

5/3 RACE Kit,

Cat. No. 03 353 621 001

Kit for 10 reactions

What this Product Does ........................................................................................................ 3

How To Use this Product ...................................................................................................... 5

Before You Begin

Experimental Protocol .......................................................................................................... 9

Experimental Protocol for 5 RACE

Additional Information on this Product........................................................................... 23

1. What this Product Does

What this Product Does

PCR Anchor Primer

1. What this Product Does

For PCR amplification of poly(A)-tailed first-strand cDNA, the following

2. How To Use this Product

How To Use this Product

Before You Begin

At least two antisense gene-specific primers are needed.

2. How To Use this Product

Control of cDNA synthesis: neo3/for

2. How To Use this Product

A gene-specific forward primer SP5 is required. Efficient and specific PCR

First-strand cDNA synthesis is initiated at the poly(A)+ tail of the neo-RNA

Oligo(dT)-Anchor Primer 1,040 bp

Amplification of cDNA neo3/for

2. How To Use this Product

Experimental Protocol for 5 RACE

cDNA Synthesis Buffer (Vial 1)

Deoxynucleotide Mixture (Vial 3)

cDNA synthesis primer SP1 (12.5 M)

poly(A) RNA or total RNA 0.2 2 g

For control reaction:

Transcriptor Reverse Transcriptase (vial 2)

Mix the reaction mixture and spin down briefly.

First-Strand cDNA Synthesis

Protocol for the Purification of cDNA

Centrifuge for 30 sec at 6,000 to 8,000 g in an Eppendorf centrifuge.

Remove the Filter Tube from the Collection Tube.

Centrifuge for 30 sec at 6,000 to 8,000 g in an Eppendorf centrifuge.

Centrifuge at least 2 min at maximum speed (approximately 13,000

Remove the Filter Tube from the Collection Tube.

Insert the Filter Tube into a sterile 1.5 ml microcentrifuge tube.

The microcentrifuge tube contains the eluted cDNA.

Reaction Buffer, 10x conc. (Vial 5)

Poly(A) Tailing of First-Strand cDNA

Mix the reaction mixture and spin down briefly.

Pipet the following into a sterile microcentrifuge tube on ice:

Oligo dT-Anchor Primer (Vial 8)

Specific primer SP2 (12.5 M)

PCR Amplification of dA-Tailed cDNA

Deoxynucleotide Mixture (Vial 3) 1 l

Expand High Fidelity Buffer

Mix the reaction mixture and spin down briefly.

Annealing temperature depends on the melting temperature of the primer used.

you end up with enough PCR product,

you used a cDNA for rare messages, you need

Diluted or undiluted PCR