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Sequencing Sample Prep
and
Gene Expression Analysis
DNA-Seq
Nextera™ DNA Sample Prep Kits
Sequencer-ready libraries in under 2 hours, from only nanograms of DNA
Nextera technology simultaneously fragments and tags DNA in a single-tube reaction,
and offers significant advantages over current library preparation methods:
• Sequencer-ready libraries in under 2 hours.
• Incorporate platform-specific tags and optional barcodes.
• Library complexity, coverage, accuracy, and bias comparable to control libraries
prepared using mechanical and other enzyme-based fragmentation methods.
• Validated on multiple second-generation sequencing platforms, including Roche
454™ and Illumina® GAII and HiSeq™ 2000.
Nextera libraries have been successfully sequenced with numerous sample types,
including:
•
•
•
•
•
•
Human genomic DNA
E. coli genomic DNA Soy genomic DNA HIV-1 cDNA amplicons
Drosophila genomic DNA
Plasmids
•
•
•
•
•
Fosmid clones
cDNA
LR-PCR amplicons
Phage DNA
and more
Table 1. Comparison of conventional and Nextera™ library preparation workflows.
Processing Step
Fragmentation
Standard Method (~µg)
✓ (15-30 min)
Collection
✓ (15 min)
Concentration
✓ (15 min)
Size Selection
✓ (60 min)
End-Repair
✓ (60 min)
Clean-Up
✓ (15 min)
A-Tailing
+/- (30 min)
Adaptor Ligation
✓ (60 min)
Clean-Up
Library Enrichment
Total Time:
Nextera Method (<50 ng)
Add Nextera Enzyme Mix
(5 min)
✓ (15 min)
(15 min)
Variable (~60 min)
PCR (~60 min)
~6 Hours
<2 Hours
Blue color indicates steps where the sample is transferred to another tube.
2
www.epicentre.com
DNA-Seq
Illumina now sells and supports a new version of Nextera
kits. These new kits offer several advantages over the
current Epicentre kits, which will be discontinued effective
December 31, 2011.
Cat. # Quantity
Nextera™ DNA Sample Prep Kit (Illumina®-Compatible) GA09115
GA091120
GA0911-50
GA0911-96
5 Reactions
20 Reactions
50 Reactions
96 Reactions
Nextera™ DNA Sample Prep Kit (Roche Titanium-Compatible)
NT09115
5 reactions
NT091120
20 reactions
NT0911-50
50 reactions
NT0911-96
96 reactions
Contents: Nextera Enzyme Mix (Illumina®-compatible), 5X Nextera Reaction Buffer (LMW), 5X Nextera
Reaction Buffer (HMW), 50X Nextera Primer Cocktail (Illumina®-compatible), 50X Nextera Adaptor 2
(Illumina®-compatible), 200X Nextera Read 1 Primer, 200X Nextera Read 2 Primer, 200X Nextera
Index Read Primer, Nextera Control DNA, and 2X Nextera PCR Buffer.
Nextera™ Barcodes (optional)
Illumina®-Compatible
GABC0950
12 Bar Codes
Roche Titanium-Compatible
NTBC0950
12 Bar Codes
Note: The barcoding kits are optional. Each kit contains 12 barcodes, and each barcode can be used to
prepare up to 50 barcoded libraries.
Additional Nextera Kits:
Nextera™ PCR Enzyme
EM091120
20 Reactions
EM091150
50 Reactions
EM0911-96
96 Reactions
Important: Nextera PCR Enzyme must be purchased separately when using any of the Nextera DNA
Sample Prep kits.
[email protected] • (800) 284-8474
3
DNA-Seq
End-It™ DNA End-Repair Kit
Repair DNA ends for adaptor ligation
The End-It DNA End-Repair Kit converts DNA containing damaged or incompatible 5′and/or 3′-protruding ends to 5′-phosphorylated, blunt-ended DNA. The end-repaired
DNA can be used for ligation into a cloning vector or ligation of next-generation
sequencing adaptors, using the Fast-Link™ DNA Ligation Kit. The high specific activity
of the End-Repair Enzyme Mix provides complete conversion of protruding ends to
5′-phosphorylated, blunt-ended DNA.
The End-It DNA End-Repair Kit contains reagents for 20 or 50 end-repair reactions (repair
of up to 100 µg or 250 µg of genomic DNA, respectively).
Cat. # Quantity
End-It™ DNA End-Repair Kit
ER0720
ER81050
Contents: End-Repair Enzyme Mix, End-Repair 10X Buffer, dNTP Solution, ATP.
20 Reactions
50 Reactions
Fast-Link™ DNA Ligation Kit
Ligate next-generation sequencing adaptors to DNA
The Fast-Link DNA Ligation Kit uses a high-quality ligase (Fast-Link T4 DNA Ligase),
to provide extremely rapid, high-efficiency DNA ligation. Cohesive-end ligations
can be performed in 5 minutes at room temperature. In contrast to other ligases,
it is not necessary to desalt Fast-Link ligation reactions prior to transformation of
electrocompetent or chemically competent cells. The Fast-Link Kit can be used for
routine and high-throughput DNA cloning.
Cat. # Quantity
Fast-Link™ DNA Ligation Kit
LK11025
LK0750H
LK6201H
Contents: Fast-Link DNA Ligase, Fast-Link 10X Ligation Buffer, 10 mM ATP.
25 Ligations
50 Ligations
100 Ligations
4
www.epicentre.com
DNA-Seq
CircLigase™ and CircLigase II ssDNA Ligases
Circularize ssDNA templates
CircLigase ssDNA Ligase* is a thermostable ATP-dependent ligase that catalyzes
intramolecular ligation (i.e., circularization) of ssDNA templates having a 5′-phosphate
and a 3′-hydroxyl group. In contrast to other ligases, CircLigase ssDNA Ligase ligates ends
of ssDNA in the absence of a complementary sequence. The enzyme is therefore useful
for making circular ssDNA molecules from linear ssDNA. Circular ssDNA molecules can be
used as substrates for rolling-circle replication or rolling-circle transcription.
CircLigase II ssDNA Ligase* has greater activity and a new Reaction Buffer for improved
ligation efficiency.
Unit Definition: One unit of CircLigase enzyme converts 1 pmol of a linear
5′-phosphorylated CircLigase Standard 55-mer Oligo into an Exonuclease I–resistant
circular form in 1 hour at 60°C under standard assay conditions.
*Covered by intellectual property rights licensed to Epicentre.
Cat. # Quantity
CircLigase™ ssDNA Ligase
CL4111K
1,000 Units
CL4115K
5,000 Units
Contents: CircLigase ssDNA Ligase, CircLigase 10X Reaction Buffer, 1 mM ATP, 50 mM MnCl2,
CircLigase ssDNA Control Oligo, Sterile Water.
CircLigase™ II ssDNA Ligase
CL9021K
CL9025K
Contents: CircLigase II ssDNA Ligase, CircLigase II 10X Reaction Buffer, 50 mM MnCl2,
CircLigase ssDNA Control Oligo, Betaine, Sterile Water.
1,000 Units
5,000 Units
DisplaceAce™ DNA Polymerase
Rapid DNA sequencing of GC-rich regions
DisplaceAce DNA Polymerase* is a recombinant DNA polymerase modified to remove
5′→3′ exonuclease activity. It has strong strand-displacing DNA polymerase activity,
similar to that of Bacillus DNA polymerases. The DNA-dependent DNA polymerase
activity is optimal at approximately 65°C. It also has RNA-dependent DNA polymerase
activity. The enzyme can be inactivated by incubation at 80°C for 20 minutes.
Unit Definition: One unit converts 10 nmol of dNTPs into acid-insoluble material in
30 minutes at 65°C under standard assay conditions.
*Covered by issued and/or pending patents.
Cat. # DisplaceAce™ DNA Polymerase
D090710K
[email protected] • (800) 284-8474
Concentration
Quantity
100U/ul 10,000 Units
5
DNA-Seq
Plasmid-Safe™ ATP-Dependent DNase
Remove bacterial chromosomal DNA from plasmid preps
Plasmid-Safe ATP-Dependent DNase selectively removes contaminating bacterial
chromosomal DNA from plasmid, cosmid, fosmid, and BAC clones or vector preparations.
Such preparations are frequently contaminated with fragments of bacterial genomic
DNA generated during alkaline lysis. Other purification options, such as spin-columns
or even CsCl centrifugation, do not effectively remove these contaminants and require
further purification steps.
Plasmid-Safe ATP-Dependent DNase digests linear dsDNA to deoxynucleotides at slightly
alkaline pH and, with lower efficiency, closed-circular and linear ssDNA. The enzyme has
no activity on nicked or closed-circular dsDNA or supercoiled DNA.
Unit Definition: One unit of Plasmid-Safe DNase converts 1 nmol of deoxynucleotides
in linear T7 DNA into an acid-soluble form in 30 minutes at 37°C under standard assay
conditions. Three units will digest 1 µg of linear T7 DNA in 30 minutes at 37°C.
Cat. # Concentration
Quantity
Plasmid-Safe™ ATP-Dependent DNase
E3101K
10 U/µl E3105K
10 U/µl E3110K
10 U/µl Includes Plasmid-Safe 10X Reaction Buffer and 25-mM ATP Solution.
1,000 Units
5,000 Units
10,000 Units
Proteinase K
Remove protein after selection in ChIP protocols
Proteinase K is used to digest protein and remove contamination from preparations of
nucleic acid. The addition of Proteinase K to nucleic acid preparations rapidly inactivates
nucleases that might otherwise degrade the DNA or RNA during purification. Proteinase K
remains active under denaturing conditions, such as the presence of SDS or urea,
chelating agents (EDTA, sulfhydryl reagents), and trypsin or chymotrypsin inhibitors.
Cat. # Proteinase K
MPRK092
6
Concentration
Quantity
50 µg/µl 2 ml
www.epicentre.com
DNA-Seq
Exonuclease III, E. coli
Produce nested deletions for site-directed mutagenesis
Exonuclease III digests ds DNA in a 3′→5′ direction from a nick, a blunt end, or
3′-recessed end, producing stretches of ssDNA on the opposite strand. Under defined
reaction conditions, DNA degradation by Exonuclease III proceeds at a uniform rate
yielding predictable and reproducible digestion results.
Exonuclease III is not active on 3′-protruding ends of four bases or more in length,
ssDNA, or on thioester-linked nucleotides.The enzyme also has intrinsic RNase H, 3′-DNA
phosphatase, and apurinic DNA endonuclease activities.
Unit Definition: One unit of Exonuclease III catalyzes the release of 1 nmol of acidsoluble nucleotides from ds calf thymus DNA in 30 minutes at 37°C under standard assay
conditions.
Cat. # Exonuclease III, E.coli
EX4405K
EX4425K
Includes 10X Reaction Buffer.
Concentration
Quantity
200 U/µl
200 U/µl
5,000 Units
25,000 Units
Exonuclease I, E. coli
Remove excess ssDNA primers after amplification
Exonuclease I digests ssDNA in a 3′→5′ direction, but does not digest dsDNA. Although
it requires the presence of magnesium and a free 3′-hydroxyl terminus for activity, it is
active under a wide variety of buffer conditions and can be added directly into most
reaction mixes. Exonuclease I can be heat-inactivated by incubation at 80°C for 15
minutes.
Unit Definition: One unit of Exonuclease I catalyzes the release of 10 nmol of acidsoluble nucleotides from heat-denatured calf thymus DNA in 30 minutes at 37°C under
standard assay conditions.
Cat. # Exonuclease I, E. coli
X40501K
X40505K
X40520K
[email protected] • (800) 284-8474
Concentration
Quantity
20 U/µl
20 U/µl
20 U/µl
1,000 Units
5,000 Units
20,000 Units
7
rRNA Removal
RiboZero™ rRNA Removal Kits
Remove >99% of the rRNA from a total RNA sample
The Ribo-Zero rRNA Removal Kits remove more ribosomal RNA (rRNA) from total RNA
preparations than any other method. The kits are ideal for preparing RNA samples for
whole-transcriptome RNA-Seq, random-primed cDNA synthesis, and other RNA analysis
applications.
•
•
•
•
Suitable for both intact and partially degraded RNA.
Recovers mRNA and small and large ncRNAs for whole-transcriptome RNA-Seq.
Single-pass, 90- to 120-minute process.
Recovers all fragments of nonribosomal RNAs in a partially degraded RNA sample.
Figure 1. Summary of RNA-Seq data after Ribo-Zero™ treatment.
Total RNA samples were
processed using the indicated
method of rRNA removal or
poly(A) enrichment. RNA-Seq
libraries were prepared using the
ScriptSeq™ Kit and sequenced on
and Illumina® GAIIx sequencer.
UHRR, Universal Human
Reference RNA; BrRR, Brain
Reference RNA; FFPE, human
breast tumor.
Cat. # Quantity
Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat)
RZH1046
RZH110424
6 Reactions
24 Reactions
Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat) Low Input
RZH1086
6 Reactions
Ribo-Zero™ rRNA Removal Kit (Meta-Bacteria)
RZMB11086
6 Reactions
Ribo-Zero™ rRNA Removal Kit (Gram-Negative Bacteria)
RZNB1056
6 Reactions
Ribo-Zero™ rRNA Removal Kit (Gram-Positive Bacteria)
RZPB10106
6 Reactions
Ribo-Zero™ rRNA Removal Kit (Plant Leaf)
RZPL11016
6 Reactions
Ribo-Zero™ rRNA Removal Kit (Plant Seed/Root)
RZSR11036
6 Reactions
8
www.epicentre.com
rRNA Removal
RiboZero™ Gold Kit (Human/Mouse/Rat)
Remove cytoplasmic and mitochondrial rRNA
The Ribo-Zero Gold Kit (Human/Mouse/Rat) is an enhanced version of Epicentre’s popular
Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat). The Ribo-Zero Gold Kit removes both
cytoplasmic (nuclear-encoded) rRNA and mitochondrial rRNA from 1 to 5 µg of human,
mouse, or rat total RNA preparations. The resulting rRNA-depleted RNA is suitable for
RNA-Seq (Fig. 1), random-primed cDNA synthesis, and other RNA analysis applications.
•
•
•
•
Suitable for both intact and partially degraded RNA.
Recovers mRNA and small and large ncRNAs for whole-transcriptome RNA-Seq.
Single-pass, 90- to 120-minute process.
Recovers all fragments of nonribosomal RNAs in a partially degraded RNA sample.
Figure 1. Profiles of RNA-seq libraries prepared after treatment with the
Ribo-Zero™ (A) and Ribo-Zero™ Gold (B) Kits.
A
B
0.52%
0.02%
0.24%
0.05%
0.31%
Aligning exomic
6.72%
Partial exomic
24.04%
31.94%
27.40%
28.10%
Intronic
Genomic
4.48%
16S mtrRNA
12.45%
32.05%
12S mtrRNA
31.68%
28S rRNA
Total RNA from MCF-7 cells was treated with either the standard Ribo-Zero Kit or the RiboZero Gold Kit, and RNA-Seq libraries were prepared using the ScriptSeq Kit. Libraries were
sequenced on Illumina® GAII and HiSeq 2000 sequencers. Data courtesy Vladimir Benes and
Jonathon Blake, EMBL GeneCore, Heidelberg, Germany.
Cat. # Quantity
Ribo-Zero™ Gold Kit (Human/Mouse/Rat)
RZHM11106 6 Reactions
[email protected] • (800) 284-8474
9
mRNA-Seq Sample Prep
ScriptSeq™ mRNA-Seq Library Preparation Kits
Directional mRNA-Seq libraries in 3 hours
The ScriptSeq mRNA-Seq Library Preparation Kits* use a unique terminal-tagging
technology* that simplifies the preparation of directional, paired-end libraries for
Illumina® sequencing. The kits are suitable for any animal, plant, or bacterial species.
•
•
•
•
•
Start with 50 ng or less of rRNA-depleted or poly(A)-enriched RNA.
Cluster-ready, directional libraries in about 3 hours without adaptor ligation.
Use Illumina-compatible barcodes (available separately) or user-defined barcodes.
Libraries demonstrate high concordance with MAQC qPCR data.
High percentage of mapped reads from intact, partially degraded, and FFPE RNA
samples.
*Covered by issued and/or pending patents.
Figure 1. Correlation of gene expression.
A. Intact UHRR and BrRR
B. Fragmented UHRR and BrRR
Correlation between data obtained from ScriptSeq™ libraries and corresponding MAQC qPCR
data. A) Libraries prepared from rRNA-depleted, intact UHRR and BrRR. B) Libraries prepared
from partially fragmented, rRNA-depleted UHRR and BrRR. UHRR, Universal Human Reference
RNA; BrRR, Brain Reference RNA.
Cat. # Quantity
ScriptSeq™ mRNA-Seq Library Preparation Kit (Illumina®-Compatible)
SS10906
SS10924
6 Reactions
24 Reactions
RNA-Seq Barcode Primers (Illumina®-Compatible)
RSBC10948
Set of 12 Illumina®-compatible barcodes.
10
48 Reactions each
www.epicentre.com
Small RNA-Seq Sample Prep
ScriptMiner™ Small RNA-Seq Library Preparation Kits
(Illumina®-Compatible)
Sequence the entire small-RNA transcriptome
The ScriptMiner Small RNA-Seq Library Preparation Kits (Illumina®-compatible) include
Tobacco Acid Pyrophosphatase (TAP) to enable capture of the entire small-RNA
transcriptome. Total RNA (1-5 µg) or size-selected RNA (100 pg) can be used as starting
material.
• Single-day procedure.
• Greatly reduced level of adaptor-dimer in the sequencing library.
• Prepare libraries from miRNA and, optionally, from small 5′-capped and small
5′-triphosphorylated RNAs.
• Strand-specific tagging of the RNA enables directional sequencing.
Figure 1. A ScriptMiner™ library captures the entire small-RNA transcriptome.
The optional TAP treatment in the ScriptMiner procedure enables the user to prepare
libraries from small 5′-monophosphorylated RNAs, as well as small 5′-capped and small
5′-triphosphorylated RNAs that are not captured by conventional small RNA-seq library prep
methods.
Cat. # Quantity
ScriptMiner™ Small RNA-Seq Library Preparation Kit (SinglePlex; Illumina®-Compatible)
SMSP10908
8 Reactions
Generates nonbarcoded, Illumina®-compatible libraries.
ScriptMiner™ Small RNA-Seq Library Preparation Kit (MultiPlex; Illumina®-Compatible)
SMMP101212
12 Reactions
Generates libraries containing Illumina®-compatible or user-defined barcodes. Illumina®-compatible
barcodes are sold separately.
[email protected] • (800) 284-8474
11
RNA-Seq
Tobacco Acid Pyrophosphatase
Remove the 5′ cap from mRNA for RNA-Seq
Tobacco Acid Pyrophosphatase (TAP) hydrolyzes the phosphoric acid anhydride bonds in
the triphosphate bridge of the cap structure found in most eukaryotic mRNA, releasing
the cap nucleoside and generating a 5′-monophosphorylated terminus on the RNA
molecule. The resulting “decapped” 5′-monophosphorylated terminus may be ligated to
a 3′-hydroxyl terminus using T4 RNA Ligase or dephosphorylated using APex™ HeatLabile Alkaline Phosphatase. Similarly, TAP digests the triphosphate group at the 5′ end
of prokaryotic transcripts, generating an RNA molecule with a 5′-monophosphorylated
terminus.
Unit Definition: One unit of TAP releases 1 nmol of inorganic phosphate from m7GpppG
in 30 minutes at 37°C under standard assay conditions.
Cat. # Tobacco Acid Pyrophosphatase (TAP)
T81050
T19050
T19100
T19250
T19500
Includes 10X Reaction Buffer.
Concentration
Quantity
5 U/µl
10 U/µl
10 U/µl
10 U/µl
10 U/µl
50 Units
50 Units
100 Units
250 Units
500 Units
Poly(A) Polymerase Tailing Kit
Add poly(A) tails to RNA for RNA-Seq
Poly(A) Polymerase uses ATP as a substrate for template-independent addition of
adenosine monophosphate to the 3′-hydroxyl termini of RNA molecules. The Poly(A)
Polymerase Tailing Kit provides the enzyme and other reagents for quickly and easily
adding a poly(A) tail to the 3′ end of any RNA.
Unit Definition: One unit of Poly(A) Polymerase catalyzes the incorporation of 1 nmol of
AMP into acid-insoluble form in 10 minutes at 37°C under standard assay conditions.
Cat. # Concentration
Quantity
Poly(A) Polymerase Tailing Kit
PAP5104H
50 Reactions Contents: Poly(A) Polymerase, 10X Reaction Buffer, 10 mM ATP, Sterile RNase-Free Water.
12
400 Units
www.epicentre.com
RNA-Seq
APex™ Heat-Labile Alkaline Phosphatase
Dephosphorylate DNA or RNA for sequencing library prep
APex Heat-Labile Alkaline Phosphatase is an innovative enzyme preparation with
improved performance over other available thermolabile alkaline phosphatases.
APex Phosphatase removes the 5′ phosphate from all types of DNA ends, including
5′-protruding, blunt, and 5′-recessed ends, and from RNA ends. The enzyme is irreversibly
heat-inactivated by incubation at 70°C for 5 minutes.
Unit Definition: One microliter of APex Heat-Labile Alkaline Phosphatase
dephosphorylates 1 µg of pUC19 vector DNA digested with Hind III (5′-protruding ends),
Hinc II (blunt ends), or Pst I (5′-recessed ends) in 10 minutes at 37°C.
Cat. # Concentration
Quantity
APex™ Heat-Labile Alkaline Phosphatase
AP49010
AP49050
AP49100
1 Reaction/µl
1 Reaction/µl
1 Reaction/µl
10 Reactions
50 Reactions
100 Reactions
RNA 5′ Polyphosphatase
Convert 5’-triphosphorylated RNA to 5’-monophosphorylated RNA for RNA-Seq
RNA 5′ Polyphosphatase* is a Mg2+-independent phosphohydrolase discovered and
characterized at Epicentre. The enzyme sequentially removes the γ and β phosphates
from 5′-triphosphorylated RNA (such as primary RNA transcripts):
5′ pppN—OH 3′ → 5′ pN—OH 3′ + 2 Pi
RNAs with a 5′-diphosphorylated end are also converted to 5′-monophosphorylated RNA
by RNA 5′ Polyphosphatase: 5′ ppN—OH 3′ → 5′ pN—OH 3′ + Pi
RNA Polyphosphatase has no activity on RNA with a 5′ cap (e.g., 5′ m7GpppN—OH 3′),
or a 5′-monophosphorylated end (5′ pN—OH 3′). However, both NTPs and dNTPs are
substrates for the enzyme, yielding the corresponding NMPs and dNMPs + inorganic
phosphate: (d)NTP → (d)NMP + 2Pi
Unit Definition: One unit of RNA 5′ Polyphosphatase releases 1 nmol of inorganic
phosphate from ATP in 1 hour at 37°C under standard assay conditions.
*Covered by issued and/or pending patents.
Cat. # RNA 5 Polyphosphatase
RP8092H
Includes 10X Reaction Buffer.
[email protected] • (800) 284-8474
Concentration
Quantity
20 U/µl
200 Units
13
RNA-Seq
T4 RNA Ligase
Prepare 5’-tagged RNA for RNA-Seq
T4 RNA Ligase catalyzes the formation of a phosphodiester bond between a
5′-phosphoryl-terminated nucleic acid donor and a 3′-hydroxyl-terminated nucleic
acid acceptor in a template-independent manner. The enzyme is ATP-dependent, and
is active on a broad range of substrates including RNA, DNA, oligoribonucleotides,
oligodeoxyribonucleotides, as well as numerous nucleotide derivatives.
Unit Definition: One unit of T4 RNA Ligase catalyzes the conversion of 1 nmol of
5′-phosphoryl termini in poly(A)18 into a phosphatase-resistant form in 30 minutes at 37°C
under standard assay conditions.
Cat. # T4 RNA Ligase LR5010
LR5025
Includes 10X Reaction Buffer and a 10-mM ATP Solution.
Concentration Quantity
5 U/µl
5 U/µl
1,000 Units
2,500 Units
T4 RNA Ligase 2, Deletion Mutant
Tag small RNAs for RNA-Seq
T4 RNA Ligase 2, Deletion Mutant, also known as T4Rnl2(1-249), is used to ligate singlestranded adenylated DNA or RNA oligonucleotides to small RNAs in the absence of
ATP. The ligation products can be used for cloning or next-generation RNA sequencing.
Performing the ligation reaction in the absence of ATP prevents circularization and other
undesirable bimolecular reactions.
Unit Definition: One unit is the amount of enzyme required to give 50% ligation of a
22-mer RNA to the preadenylated end of a 17-mer DNA when both oligos are annealed
to a complementary 39-mer DNA strand in 30 minutes at 37°C under standard assay
conditions.
Cat. # T4 RNA Ligase 2, Deletion Mutant
LR2D1132K
LR2D11310K
14
Concentration Quantity
200 U/µl
200 U/µl
2,000 Units
10,000 Units
www.epicentre.com
Illumina® BeadChip® Target Prep
TargetAmp™ Labeling Kits for Illumina® Expression
BeadChip® Arrays
Low-input RNA amplification and labeling
TargetAmp-Nano Target Labeling Kit for Illumina Expression BeadChip
• Produce microgram amounts of biotin-aRNA from 25 ng to 500 ng of RNA.
• Perform the 6-hour TargetAmp-Nano Kit reaction and begin BeadChip hybridization
the same day.
TargetAmp-Pico Target Labeling Kit for Illumina Expression BeadChip
• Produce microgram amounts of biotin-aRNA from 50 pg to 500 pg of RNA.
• Ideal for very small samples, such as laser-capture microdissected (LCM) samples.
Figure 1. Genes detected using target labeled with the
TargetAmp™-Nano and TargetAmp™-Pico kits.
Number
of genes
detected
Number
of genes
detected
20000
18000
16000
TargetAmp™-Pico Kit
TargetAmp™-Nano Kit
14000
12000
10000
8000
6000
4000
2000
0
50 pg
50 pg 100 pg 100 pg 100 ng 100 ng 500 ng 500 ng
Liver Muscle Liver
Muscle Liver Muscle Ovary Testicle
RNAsource
source and
amount
RNA
and
amount
Target RNA from the indicated source was labeled using either the TargetAmp-Nano or
TargetAmp Pico kits. The target RNA was hybridized to either the Mouse Ref-8 Expression
BeadChip or HumanHT-12 v4 Expression BeadChip (Illumina).
Cat. # Quantity
TargetAmp™-Nano Target Labeling Kit for Illumina® Expression BeadChip®
TAN07924
TAN091096
For use with 25-500 ng of RNA
24 Reactions
96 Reactions
TargetAmp™-Pico Target Labeling Kit for Illumina® Expression BeadChip®
TAP110610
For use with 50-500 pg of RNA
10 Reactions
Additional TargetAmp kits are available. Visit www.epicentre.com for information.
[email protected] • (800) 284-8474
15
Single-Cell qRT-PCR Analysis
MessageBOOSTER™ cDNA Synthesis from
Cell Lysates Kit
Sensitive qRT-PCR from one cell…without purifying RNA!
A MessageBOOSTER cDNA Synthesis from Cell Lysates Kit reaction amplifies the poly(A)
RNA directly from a cell lysate. and then converts the amplified RNA to cDNA that is ready
for qPCR. There is no need to isolate total RNA.
• Low-abundance transcripts in a single cell are readily detected.
• Hundreds of sensitive qRT-PCRs obtained from as little as a single cell. cDNA can be
archived for future use.
• Linear RNA amplification process preserves the gene expression profile of the cell(s).
Relative Fluorescence Units
Figure 1. Sensitivity of the MessageBOOSTER™ Kit.
1:100
1:1,000
1:10
undiluted
Cycle
qPCR was performed using undiluted (red), 1:10 diluted (green), 1:100 diluted (blue), and
1:1,000 diluted (purple) cDNA produced from a lysate of a single NRK cell. The low-abundance
PBGD transcript was readily detected.
Cat. # Quantity
MessageBOOSTER™ cDNA Synthesis Kit from Cell Lysates Kit
MBCL90310
10 Reactions
16
www.epicentre.com
Rapid Extraction of PCR-Ready DNA
QuickExtract™ DNA Extraction Kits
Rapidly prepare PCR-ready DNA from various sample types
QuickExtract products offer a rapid and efficient method for extracting genomic DNA
from virtually any sample for PCR-based assays. Most samples can be processed in
8 minutes with only two sequential heating steps.
• Fast—PCR-ready DNA in 3-8 minutes, ideal for high-throughput workflows.
• Safe—No organic extraction.
• Efficient—No columns, transfers, or sample loss.
Figure 1. FailSafe™ PCR amplification of extracted DNA
M
1
2
3
4
5
6
Buccal cells were extracted using the BuccalAmp™
DNA Extraction Kit, and all other samples with
QuickExtract™ DNA Extraction Solution. PCR was
performed using primers to amplify the regions
indicated: Lanes 1-3, human β-globin (human
buccal cells, HeLa cells, and human hair follicle,
respectively); lane 4, transgenic mouse GAPDH
(mouse tail snip); lane 5, E. coli 16S ribosomal RNA
gene (bacteria); lane 6, transgenic SV40 T antigen
(mouse tail snip).
Figure 2. PCR amplification of FFPE DNA
M 1 2 3 4 5 6 7 M
DNA was extracted from a slide-mounted, FFPEpreserved human skeletal muscle tissue section with the
QuickExtract™ FFPE DNA Extraction Kit. Two microliters of
undiluted, extracted DNA was amplified with primers for
three different loci: tumor protein 53 (TP53), dystrophin
(DMD), and tumor necrosis factor (TNF). The products were
separated on a 3% agarose gel and were visualized with
SYBR® Gold. Lane M, 100-bp DNA ladder; lane 1, exon 2 of
TP53; lane 2, exon 3 of TP53; lane 3, exon 11 of TP53; lane 4,
exon 6 of DMD; lane 5, exon 50 of DMD; lane 6, exon 3 of
DMD; lane 7, exon 4 of TNF.
Cat. # Quantity
QuickExtract™ DNA Extraction Solution
QE0905T
QE09050
5 ml
50 ml
[email protected] • (800) 284-8474
17
Rapid Extraction of Nucleic Acids
QuickExtract™ Family of Products
Products
Time
Applications
Samples Tested
QuickExtract™ DNA
Extraction Solution
(QE0905T, QE09050)
3-8 min
Genotyping, genetic
studies, identity testing,
viral/microbial screening
Hair follicles, feathers (quillend cells), tissue-culture cells,
buccal cells, zebrafish (organs,
scales), mouse tail snips
BuccalAmp™ DNA
Extraction Kits
(BQ0901SCR, BQ0908SCR,
BQ0916SCR)
3-8 min
Genotyping, genetic
studies, identity testing
Buccal cells from human or
animal subjects. Catch-All™
Buccal Swabs included.
QuickExtract™ RNA
Extraction Kit
(QER09015, QER090150)
6 min
RT-PCR
Cultured cells: human, mouse,
rat, E. coli, S. aureus
QuickExtract™ Plant DNA
Extraction Solution
(QEP80705, QEP70750)
8 min
PCR, e.g., GMO testing
Arabidopsis, barley, maize,
emmer, pepper, rice, spelt,
spinach, soybeans, wheat
QuickExtract™ Seed DNA
Extraction Solution
(QES08095T, QES080950)
8 min
PCR, e.g., GMO testing
Apple, cotton, sunflower,
tomato, barley, maize, oats,
rice, rye, wheat
QuickExtract™ Bacterial
DNA Extraction Kit
(QEB0905T, QEB09050)
15 min
PCR, restriction digests,
PFGE, optical mapping
Gram-positive:
Bacillus subtilis
Bifidobacterium spp
Brevibacterium linens
Clostridium perfringens
Lactobacillious plantarum
Listeria monocytogenes
Staphylococcus equorum
Streptococcus agalactiae
Streptococcus pyrogenes
Streptococcus thermophilus
Gram-negative:
E. coli
Salmonella enterica
Salmonella typhimurium
Vibrio gazogenes
QuickExtract™ FFPE DNA
Extraction Kit
(QEF81805, QEF81050)
62 min
Microsatellite,
single-nucleotide
polymorphisms (SNP),
tumor heterogeneity
studies, copy number
variations (CNV),
methylation analysis, short
tandem repeats (STR)
Formalin-fixed, paraffinembedded human tissue
samples
QuickExtract™ FFPE RNA
Extraction Kit
(QFR82805, QFR82050)
32 min
RT-PCR
Formalin-fixed, paraffinembedded human tissue
samples
For more information, see: www.epicentre.com/quickextract
18
www.epicentre.com
Other Reagents
DNA and RNA Purification
Cat. # Quantity
MasterPure™ Complete DNA and RNA Purification Kit
MC89010
MC85200
5 DNA/10 RNA Purifications
200 DNA/100 RNA Purifications
MasterPure™ DNA Purification Kit for Blood Version II
MB711740
MB711400
For 40 ml of whole blood
For 400 ml of whole blood
MasterPure™ RNA Purification Kit
MCR85102
100 Purifications
PCR
Cat. # Concentration Quantity
MasterAmp™ Extra-Long PCR Kit
MHF9220
50 Reactions
MasterAmp™ Extra-Long DNA Polymerase Mix (Enzyme Mix Only)
QU92125
2.5 U/µl
QU92500
2.5 U/µl
QU9201K
2.5 U/µl
125 Units
500 Units
1,000 Units
Other Enzymes
Cat. # Concentration Quantity
1 µg/µl (1,000 U/µl)
0.1 µg/µl (100 U/µl)
10 µg (10,000 Units)
10 µg (10,000 Units)
RepliPHI™ Phi29 Reagent Set
RH031110
1 µg/µl (1,000 U/µl)
RH040210
0.1 µg/µl (100 U/µl)
Includes buffer, dNTPs, DTT, and enzyme at the indicated concentration/size.
10 µg (10,000 Units)
10 µg (10,000 Units)
RepliPHI™ Phi29 DNA Polymerase (enzyme only)
PP031010
PP040110
Exo-Minus Klenow DNA Polymerase
KL0810250
KL081001K
KL080911K
Baseline-ZERO™ DNase
DB0711K
DB0715K
RNase A
MRNA092
RiboShredder™ RNase Blend
RS12100
RS12500
[email protected] • (800) 284-8474
5 U/µl
5 U/µl
10 U/µl
1 U/ul 1 U/ul 250 Units
1,000 Units
1,000 Units
1,000 MBU
5,000 MBU
5 mg/ml
2 ml
1 U/µl
1 U/µl
100 Units
500 Units
19
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Madison, WI 53713