Superscript Firststrand Synth cDNA Manual
Transcript of Superscript Firststrand Synth cDNA Manual
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Instruction Manual
SuperScript First-Strand SynthesisSystem for RT-PCR
Catalog No. 11904-018
Version E
11 August 2003
53034
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Table of Contents
Kit Contents and Storage.......................................................................... 1
Introduction ..............................................................................................2
First-Strand cDNA Synthesis ................................................................... 5Amplification of the Target cDNA........................................................... 8
Troubleshooting...................................................................................... 11
Troubleshooting with the Control RNA .................................................13
Additional Protocols ............................................................................... 16
References ..............................................................................................20
Related Products..................................................................................... 21
Purchaser Notification ............................................................................ 23Technical Service ................................................................................... 25
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Kit Contents and Storage
KitComponents
The components of the SuperScript First-Strand Synthesis System
for RT-PCR are listed below. Components are provided in
sufficient quantities to perform a total of 50 separate reactions; each
converts from 1 ng up to 5 g of total RNA into first-strand cDNA.
The SuperScript First-Strand Synthesis System for RT-PCR is
stored at 20C.
Component Amount
Oligo(dT)12-18 (0.5 g/l) 50 l
Random hexamers (50 ng/l) 250 l
10X RT buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl ] 1 ml
25 mM MgCl2 500 l
0.1 M DTT 250 l
10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP) 250 l
SuperScript II RT (50 units/l) 50 l
RNaseOUT Recombinant Ribonuclease Inhibitor (40 units/l) 100 l
E. coli RNase H (2 units/l) 50 l
DEPC-treated water 1.2 ml
Control RNA (50 ng/l) 15 l
Control Primer A (10 M) 20 l
Control Primer B (10 M) 20 l
AdditionalMaterialsRequired
The following items are required for use with the system, but they
are not included:
DNA polymerase for PCR
Programmable thermal cycler
Autoclaved 0.2- or 0.5-ml microcentrifuge tubes or autoclaved,thin-walled PCR tubes
Automatic pipettes and tips capable of dispensing 120 l and
20200 l
Disposable gloves
Two amplification primers specific for your target mRNA
Microcentrifuge capable of generating a relative centrifugal
force of 14,000 g
37C, 42C, 65C, and 70C water baths or heat blocks
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Introduction
SystemSummary
The SuperScript First-Strand Synthesis System for RT-PCR is
optimized to synthesize first-strand cDNA from purified poly(A)+ or total
RNA. The system can be used with as little as 1 ng or as much as 5 g of
total RNA.
After synthesis, target cDNA can be amplified with specific primers by
PCR without intermediate organic extractions or ethanol precipitations, as
shown in the diagram below.
mRNA
AAAAAA
(primer) TTTTTT
First-strand synthesis
AAAAAA
TTTTTT
Removal of RNA
TTTTTT
First-strand cDNA ready for PCR
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Introduction, Continued
Isolation ofTotal RNA
One important factor for synthesis of full-length cDNA is the
isolation of intact RNA. The quality of the RNA dictates the
maximum amount of sequence information that can be converted
into cDNA. Thus, it is important to optimize the isolation of RNAand prevent introduction of RNases into the RNA preparation.
RNA can be isolated by many methods. We recommend using the
Micro-to-Midi Total RNA Purification System or TRIzol Reagent,
which yields undegraded RNA from cultured cells or whole tissue
samples. High-quality RNA can be purified from as little as 100
cells up to 1 107 cells or 200 mg of tissue.
Oligo(dT)-selection for poly(A)+ RNA is typically not necessary,
although it may improve the yield of specific cDNAs. Poly(A)+
RNA selection may also reduce the likelihood of genomic DNAcontamination. Frequently, RNA preparations contain small
amounts of genomic DNA that may be amplified along with the
target cDNA. If your application requires removal of all genomic
DNA from your RNA preparation, refer to DNase I Digestion of
RNA Preparation on page 18.
First-Strand
cDNASynthesisfrom TotalRNA
The first-strand cDNA synthesis reaction is catalyzed by
SuperScript
II Reverse Transcriptase (RT). This enzyme has beenengineered to reduce the RNase H activity (found in other RTs) that
degrades mRNA during the first-strand reaction.
Use of an RT with reduced RNase H activity results in greater full-
length cDNA synthesis and higher yields of first-strand cDNA than
obtained with other RTs. SuperScript II RT has been engineered to
retain the full DNA polymerase activity found in RNase H+ M-
MLV RT. This further improves the enzymes ability to copy long
RNA as compared to M-MLV RT or other derivatives. Because
SuperScript
II RT is not inhibited significantly by ribosomal andtransfer RNA, it may be used effectively to synthesize first- strand
cDNA from a total RNA preparation. The enzyme exhibits
increased thermal stability and may be used at temperatures up to
50C.
This system has been optimized to synthesize first-strand cDNA
from varying amounts of starting material. The SuperScript II
concentration has been lowered and RNaseOUT Recombinant
RNase Inhibitor has been added to the system as part of this
optimization process. Additionally, reaction conditions have beenmodified to further increase the sensitivity of the system.
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First-Strand cDNA Synthesis
ProcedureOverview
This procedure is designed to convert 1 ng to 5 g of total RNA or
50 to 500 ng of poly (A)+ RNA into first-strand cDNA. Please
review all the protocols before using the system.
A Control RNA is included in the system to verify performance.
Use 1 l (50 ng) of Control RNA as a template for first-strand
synthesis. Refer to Troubleshooting on page 11 for additional
information.
Continued on next page
RNA + Primer + dNTPS
65C for 5 min
Place on ice for at least 1 min
Oligo (dT)12-18 or GSP
Add all components except
1 l of SuperScript
II RT
Mix gently
Centrifuge briefly
Place tubes at 42C for 2 minAdd 1 l of SuperScript
II RT
Mix gently
42C for 50 min
70C for 15 min
Add 1 l of RNase H
Mix gently
37C for 20 min
PCR or store at 20C
Denature:
Anneal:
cDNA Synthesis:
Terminate Reaction:
Remove RNA:
25C for 10 min
Random Hexamers
Add all components except
1 l of SuperScript
II RT
Mix gently
Centrifuge briefly
Place tubes at 25C for 2 minAdd 1 l of SuperScript
II RT
Mix gently
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First-Strand cDNA Synthesis, Continued
If you have >5 g of total RNA, increase reaction volumes and
amount of SuperScript II RT proportionally.
First-StrandSynthesisUsingOligo(dT) or aGSP
1. Mix and briefly centrifuge each component before use.
2. Prepare RNA/primer mixtures in sterile 0.2- or 0.5-ml tubes
as follows:
Component Sample No RT Control Control RNA
up to 5 g total RNA n l n l
Control RNA (50 ng/l) 1 l
10 mM dNTP mix 1 l 1 l 1 l
Oligo(dT)12-18 (0.5 g/l) 1 l 1 l 1 l
or
2 M GSP 1 l 1 l
DEPC-treated water to 10 l to 10 l to 10 l
3. Incubate each sample at 65C for 5 min, then place on ice for
at least 1 min.
4. Prepare the following reaction mixture, adding eachcomponent in the indicated order. (Forn samples + 1 no RT
control + 1 Control RNA reaction,prepare the reaction mix
forn + 3 reactions.)
Component Each Rxn 4 Rxns
10X RT buffer 2 l 8 l
25 mM MgCl2 4 l 16 l
0.1 M DTT 2 l 8 l
RNaseOUT
Recombinant RNase Inhibitor 1 l 4 l5. Add 9 l of reaction mixture to each RNA/primer mixture,
mix gently, and collect by brief centrifugation.
6. Incubate at 42C for 2 min.
7. Add 1 l (50 units) of SuperScript II RT to each tube except
the no RT control, mix, and incubate at 42C for 50 min.
8. Terminate the reactions at 70C for 15 min. Chill on ice.
9. Collect the reactions by brief centrifugation. Add 1 l ofRNase H to each tube and incubate for 20 min at 37C before
proceeding to Amplification of the Target DNA on page 8.
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First-Strand cDNA Synthesis, Continued
For most RT-PCR applications, 50 ng of random hexamers per 5 g
of total RNA is adequate. Increasing hexamers to 250 ng per 5 g
of RNA may increase yield of relatively small PCR products
(
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Amplification of the Target cDNA
ProcedureOverview
The first-strand cDNA obtained in the previous procedure may be
amplified directly using PCR. Use only 10% of the first-strand
reaction (2 l) for PCR. Adding larger amounts of the first-strand
reaction may actually decrease the amount of product synthesized.PCR can use several polymerases.
Annealing and extension conditioning are primer and template
dependent, and therefore must be determined empirically.
PCR forTargets up to4 kb
Use Taq DNA Polymerase or Platinum
Taq DNA Polymerase.PlatinumTaq DNA Polymerase provides automatic hot-start
conditions for increased specificity and sensitivity.
1. Add the following to a 0.2- or 0.5-ml thin-walled PCR tube:
Volume (l)Component 1 Rxn 10 Rxns
10X PCR buffer minus Mg 5 50
50 mM MgCl2 1.5 15
10 mM dNTP mix 1 1010 M sense primer 1 10
10 M antisense primer 1 10
Taq DNA Polymerase (5 units/l) or
PlatinumTaq DNA Polymerase (5 units/l) 0.4 4
cDNA (from the first-strand reaction) 2 20
autoclaved, distilled water 38.1 381
final volume 50 500
Note: The concentration of MgCl2 listed above is approximate. Foroptimal amplification, determine the optimal MgCl2
concentration for your primers and template.
2. Mix gently and overlay with silicone oil or mineral oil if the
thermal cycler lacks a heated lid.
3. Incubate at 94C for 2 min, then perform 2040 cycles of
PCR with optimized conditions for your sample (1 min/kb
extension at 6872C).
4. Analyze 10 l of the amplified sample using agarose gelelectrophoresis.
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Amplification of the Target cDNA, Continued
Annealing and extension conditions are dependent on the primers
and template, and therefore must be determined empirically.
MaximumFidelity PCRfor Targetsup to 12 kb
PlatinumPfx DNA Polymerase possesses a proofreading 3 to 5
exonuclease activity and provides the highest fidelity of any DNA
polymerase for PCR.
1. Add the following to a 0.2- or 0.5-ml, thin-walled tube at
either ambient temperature or on ice:
Volume (l)Component 1 Rxn. 10 Rxns.
10XPfx amplification buffer 5 50
50 mM MgSO4 1 10
10 mM dNTP mix 1.5 15
10 M sense primer 1.5 15
10 M antisense primer 1.5 15
PlatinumPfx DNA Polymerase (2.5 U/l) 0.5 5
cDNA (from the first-strand reaction) 2 20
DEPC-treated water 37 370final volume 50 500
Note: For most targets 1.25 units is sufficient. When amplifying
longer targets, above 3 kb, use 2.5 units and increase
magnesium (to 1.5 mM).
Note: The concentration of MgSO4 listed above is approximate.
For optimal amplification, determine the optimal MgSO4
concentration for your primers and template.
2. Mix gently and overlay with silicone oil or mineral oil if the
thermal cycler lacks a heated lid.
3. Incubate at 94C for 2 min, then perform 2040 cycles of
PCR with optimized conditions for your sample (1 min/kb
extension time at 6872C).
4. Analyze 10 l of the amplified sample by agarose gel
electrophoresis.
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Troubleshooting
Problem Possible Cause Suggested Solution
No bands after
electrophoretic
analysis of
amplified products
Procedural error in first-
strand cDNA synthesis
Use the Control RNA to verify the efficiency of
the first-strand reaction (see the next page on
troubleshooting with the Control RNA).
RNase contamination Add Control RNA to sample to determine if
RNase is present in the first-strand reaction.
Maintain aseptic conditions to prevent RNase
contamination.
Use RNaseOUT Recombinant RNase Inhibitor
in the first-strand reaction.
Polysaccharidecoprecipitation of RNA
Precipitate RNA with lithium chloride (seepage 19) to remove polysaccharides.
Target mRNA contains
strong transcriptional
pauses
Use random hexamers instead of oligo(dT) in
the first-strand reaction.
Maintain an elevated temperature after the
annealing step (see page 16, First-strand cDNA
Synthesis of Transcripts with High-GC
Content).
Increase the temperature of first-strand reaction
(up to 50C).
Use PCR primers closer to the 3 terminus of the
target cDNA.
Too much first strand
product was used in
PCR
Use no more than 1/10 of the first-strand
reaction.
GSP was used for first-
strand synthesis
Try another GSP or switch to oligo(dT). Make
sure the GSP is the antisense sequence.
Inhibitors of RT present Remove inhibitors by ethanol precipitation of
the mRNA preparation before the first-strand
reaction. Include a 70% (v/v) ethanol wash of
the mRNA pellet.
Note: Inhibitors of RT include sodium dodecyl
sulfate (SDS), EDTA, guanidinium salts,
formamide, sodium pyrophosphate, and
spermidine.
Test for the presence of inhibitors by mixing1 g of control RNA with 1 g of sample RNA
and comparing yields of first-strand cDNA.
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Troubleshooting, Continued
Problem Possible Cause Suggested Solution
Unexpected bands
after electrophoretic
analysis
Contamination by
genomic DNA
Pretreat RNA as described in DNase I Digestion
of RNA Preparation on page 18.
Design primers that anneal to sequence in exons
on both sides of an intron or exon/exon
boundary of the mRNA to of amplified allow
differentiation between amplification of cDNA
and products potential contaminating genomic
DNA.
To test if products were derived from DNA, do
the No RT control.
Nonspecific annealing
of primers
Vary the annealing conditions. Use Platinum
Taq DNA Polymerase for automatic hot-start
PCR.
Optimize magnesium concentration for each
template and primer combination.
Primers formed dimers Design primers without complementary
sequences at the 3 ends.
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Troubleshooting with the Control RNA
Introduction The Control RNA provided with this System is an 891-bp, in vitrotranscribed RNA from the chloramphenicol acetyltransferase (CAT)
gene that has been engineered to contain a 3 poly(A) tail.
When used with Control Primers A and B in RT-PCR, the ControlRNA will result in a 500-bp product (see page 15).
First-strandcDNASynthesiswith Control
RNA
Up to 1 g of your total RNA preparation may be added to the
following mixture to evaluate the effect of this RNA on first-strand
synthesis and subsequent amplification.
1. Label two autoclaved 0.5-ml tubes A and B. Tube A
will have an addition of radioisotope to determine the
efficiency of first-strand synthesis. An aliquot from tube B
will be used for amplification.
2. Prepare the RNA/primer mixtures in sterile 0.5-ml tubes as
follows:Volume (l)
Component Tube A Tube B
Control RNA 1 1
Oligo(dT) 1 1
DEPC-treated water 7 810 mM dNTP mix 1 1
final volume 9 10
3. Incubate each sample at 65C for 5 min and place on ice for
at least 1 min. Collect by brief centrifugation and add the
following to each tube:Volume (
l)
Component Tube A Tube B
10X RT buffer 2 2
25 mM MgCl2 4 4RNaseOUT RNase Inhibitor (40 U/l) 1 1
0.1 M DTT 2 2
4. Incubate at 42C for 2 min.
5. Add 1 l of [-32P]dCTP (3,000 Ci/mmol; 10 mCi/ml) to
tube A.
6. Add 1 l (50 units) of SuperScript II RT to each tube.
Final volume will be 20
l.
7. Mix gently and collect the reaction by brief centrifugation.
8. Incubate at 42 for 50 min (continued on next page).
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Troubleshooting with the Control RNA, Continued
First-strandcDNASynthesiswith ControlRNA,Continued
9. Terminate both reactions at 70C for 15 min. Place on ice
for 1 min.
10. Collect the reaction by brief centrifugation. Add 1 l ofRNase H to tube B and incubate for 20 min at 37C. Proceed
with tube B to Amplification of the Control-Synthesized
cDNA on page 15.
11. Add 80 l of distilled water to tube A and mix gently.
12. Remove two 5-l aliquots from tube A and spot the aliquots
onto glass fiber filters. Dry one of the filters under a heat
lamp or at room temperature. This filter will be used to
determine the specific activity of the dCTP reaction.13. Wash the other filter three times, 5 min each time, with
50 ml of ice-cold, 10% (w/v) TCA containing 1% (w/v)
sodium pyrophosphate. Wash the filter once with 50 ml of
95% ethanol at room temperature for 2 min. Dry the filter
under a heat lamp or at room temperature. This filter will be
used to determine the yield of first-strand cDNA.
14. Count both filters in standard scintillant to determine the
amount of32P in the reaction, as well as the amount of32P
that was incorporated.
15. Using equation 1, determine the specific activity (SA) of the
dCTP in the first-strand reaction from the counts obtained
from the unwashed filter.
SA (cpm/pmol dCTP) = cpm/5 l
500 pmol dCTP/5 l
16. Using equation 2, determine the yield of cDNA from the
counts obtained from the washed filter and the specific
activity calculated from the unwashed filter:
Amount of cDNA (g) = (cpm) (100 l/5 l) (4 pmol dNTP/pmol dCTP)
(SA) (3,030 pmol dNTP/g cDNA)
You can assume that the yield calculated for the labeled
cDNA in tube A is equivalent to that of the unlabeled cDNA
in tube B.
17. Following first-strand cDNA synthesis using the Control
RNA, you may wish to analyze the remaining labeled cDNA
in tube A by alkaline agarose gel electrophoresis ordenaturing PAGE.
The first-strand yield using the Control RNA should be 3050%.
Electrophoretic analysis should yield a prominent 891-bp band
representing 2550% of the cDNA synthesized.
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Troubleshooting with the Control RNA, Continued
Amplificationof theControl-SynthesizedcDNA
Following first-strand cDNA synthesis from the Control RNA,
Control Primers A and B can be used in the following PCR
procedure.
Primer A (anti-sense primer): 5GAC ATG GAA GCC ATC ACA GAC3
Primer B (sense primer): 5AGA CCG TTC AGC TGG ATA TTA C3
Electrophoretic analysis of DNA products amplified using the
control primers should yield a prominent 500-bp band.
1. Perform serial dilutions with the control cDNA from tube B
with DEPC-treated or sterile, distilled water. Dilute at
1:1,000, 1:10,000, 1:100,000, and 1:1,000,000.
Note: If you assume that 1% of the total RNA is mRNA, then
50 ng of Control RNA in the first-strand reaction is
equivalent to the amount of mRNA in 5 g of total RNA.
One microliter of the 1:1,000,000 dilution of the first-strand
reaction should contain ~5,000 molecules of cDNA.
2. For each concentration, prepare a PCR mixture. Add the
following to a 0.2-ml tube sitting on ice:
Volume (l)Component 1 Rxn. 10 Rxns.
DEPC-treated water 37.6 376
10X PCR buffer minus Mg 5 50
25 mM MgCl2 3 30
10 mM dNTP mix 1 10
Control primer A (10 M) 1 10
Control primer B (10 M) 1 10
dilution of cDNA from Control RNA 1 10
Taq DNA Polymerase (5 units/l) 0.4 4
final volume 50 500
3. Mix the contents of the tube. Centrifuge briefly to collect the
reaction components.
4. Place reaction mixture in preheated (94C) thermal cycler.
Do an initial denaturation step: 94C for 2 min.
5. Perform 35 cycles of PCR:
Denature 94C for 15 sec
Anneal 58C for 30 sec
Extend 68C for 1 min
Note: If you are using a slow ramping machine (e.g., PE 480),
please follow the manufacturers suggestions.
6. Upon completion, maintain reactions at 4C.
7. Analyze 10 l of the amplified sample, using agarose gel
electrophoresis and ethidium bromide staining. A prominent
500-bp band should be visible for all four concentrations.
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Additional Protocols
First-strandcDNASynthesis ofTranscriptswith High-GCContent
High-GC content mRNAs often contain stable intrinsic secondary
structures that pose barriers to reverse transcriptase and/or primer
annealing. Problems with RT-PCR due to secondary structure of the
target mRNA often can be overcome by increasing the volume andtemperature of the RT reaction.
For templates that require cDNA synthesis temperatures above
50C, we recommend the ThermoScript RT-PCR System.
ThermoScript RT is a genetically engineered mutant of avian
retroviral reverse transcriptase with reduced RNase H activity that
supports cDNA synthesis up to 70C.
To avoid secondary RNA structure, shift the RNA/primer mix
directly from 65C to 50C and prewarm the complete 2X reaction
mix to 50C before adding it to the primer and RNA. Using athermal cycler simplifies the multiple temperature shifts and can
help prevent formation of secondary structure in RNA.
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Additional Protocols, Continued
Protocol This protocol is suitable for gene-specific or oligo(dT) primers, butnot random hexamers.
1. Mix and briefly centrifuge each component before use.
2. Prepare the RNA/primer mixture in a sterile 0.5-ml tube as
follows:
Component Sample No RT Control Control RNA
1 to 5 g total RNA n l n l
Control RNA (50 ng/l) 1 l
Oligo(dT)1218 (0.5 g/l) 1 l 1 l 1 l
or
2 M GSP 1 l 1 l 1 l
10 mM dNTP mix 2.5 l 2.5 l 2.5 l
DEPC-treated water to 25 l to 25 l to 25 l
3. Incubate each sample at 65C for 5 min and immediately
transfer to 50C.
4. Prepare the following reaction mixture, adding each
component in the indicated order. Forn samples + 1 no RT
control and 1 Control RNA reaction,prepare the reaction mix
forn + 3 reactions.
Component Each Reaction 4 Reactions
DEPC-treated water 4 l 16 l10X RT buffer 5 l 20 l
25 mM MgCl2 10 l 40 l
0.1 M DTT 5 l 20 l
RNaseOUT
Recombinant RNase Inhibitor 1 l 4 l
5. Prewarm the reaction mixture to 50C.
6. To each sample incubating at 50C, add 25 l of prewarmed
reaction mixture. Add 1 l (50 units) of SuperScript
II RT
to each tube except the no RT control, mix gently, and
incubate at 50C for 50 min.
7. Terminate the reactions at 70C for 15 min. Chill on ice.
8. Collect the reactions by brief centrifugation. Add 1 l of
RNase H to each tube and incubate for 20 min at 37C before
proceeding to PCR (page 8).
Frequently, problems associated with RT-PCR of GC-rich cDNA
are related to PCR as well as first-strand synthesis. We recommend
using PCRx Enhancer Solution, a PCR cosolvent, to facilitate
efficient amplification of GC-rich sequences.
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Additional Protocols, Continued
DNase IDigestion ofRNAPreparation
If amplification products are detected from the PCR reaction in the
absence of SuperScript II RT, it may be necessary to eliminate
residual genomic DNA from the RNA sample. After confirming the
efficiency of the first-strand synthesis reaction with the ControlRNA, use the following protocol to remove genomic DNA from the
total RNA preparation. Amplification Grade DNase I has been
extensively purified to remove trace ribonuclease activities
commonly associated with other RNase-free enzyme preparations
and does not require the addition of placental RNase inhibitor.
The following procedure requires careful pipetting of all solutions
so that the concentration of divalent metal cation (Mg2+ and Ca2+) is
controlled. Because the DNase I must be heated to 65C to
inactivate the enzyme, the concentration of free divalent metal ionsmust be low enough (
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Additional Protocols, Continued
LithiumChloridePurificationof RNAPreparation
Polysaccharides or small RNAs (tRNA and 5S RNA) that co-
precipitate with mRNA may adversely affect first-strand cDNA
synthesis. Highly purified mRNA can be recovered using the
following protocol adapted from Sambrook et. al. (1).1. To the RNA sample in DEPC-treated water, add 0.1 volume
8 M LiCl (RNase-free) and vortex. Incubate on ice for
2 hours.
2. Centrifuge at 13,000 g for 30 min at 4C.
3. Remove the supernatant, being careful not to disturb the
pellet (the pellet may be difficult to see). Dissolve the pellet
in 200 l of DEPC-treated water by drawing the pellet in
and out of a sterilized pipet tip.
4. Repeat steps 1 through 3.
5. Add 0.1 volume of 3 M sodium acetate (pH 5.2) and
2.0 volumes of absolute (100%) ethanol (20C). Place the
tube at 20C for 30 min. Centrifuge at 13,000 g for 30
min at 4C.
6. Remove the supernatant carefully. Overlay the pellet with
100 l of 70% ethanol (20C), and centrifuge at 13,000 gfor 10 min at 4C. Remove the supernatant, and air-dry the
RNA pellet at room temperature.
7. Dissolve the pellet in 10100 l of DEPC-treated water.
8. If your starting cell or tissue sample was small (1 103 to
1 105 cells), dissolve the pellet in 10 l of DEPC-treated
water and use this entire amount for first-strand synthesis. If
your starting sample was large (>1 106 cells),
spectrophotometrically determine the concentration of thepurified RNA before proceeding to page 5, First-Strand
cDNA Synthesis.
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Simms, D., Cizdziel, P.E., and Chomczynski, P. (1993)Focus
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Westfall, B., Sitaraman, K., Solus, J., Hughes, J., and Rashtchian, A. (1997)Focus
19, 46.
Westfall, B., Sitaraman, K., Berninger, M., and Mertz, L.M. (1995)Focus 17, 62.
Westfall, B., Sitaraman, K., Lee, J., Borman, J. and Rashtchian, A. (1999)Focus
21,
49.
Takagi, M., Nishioka, M., Kakihara, H., Kitabayashi, M., Inoue, H., Kawakami, B.,
Oka, M., and Imanaka, T. (1997)Appl. Environ. Microbiol. 63, 4504.
Sitaraman, K., Darfler, M., and Westfall, B. (1999)Focus 21, 10.
Nathan, M., Mertz, L., Fox, D. (1995)Focus 17, 78.
Schwabe, W., Lee, J.E., Nathan, M., Xu, R.H., Sitaraman, K., Smith, M., Potter, R.J.,Rosenthal, K., Rashtchian, A., Gerard, G.F. (1998)Focus
20, 30.
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21
Related Products
Product Size Cat. No.
Products for RNA Isolation:
FastTrack 2.0 mRNA Isolation Kit 6 reactions K1593-02
Micro-FastTrack
mRNA Isolation Kit 20 reactions K1520-02
TRIzol
Reagent 100 ml
200 ml
15596-026
15596-018
TRIzol
LS Reagent 100 ml
200 ml
10296-010
10296-028
Micro-to-Midi Total RNA Purification System 50 reactions 12183-018
Products for Analysis:
E-Gel Pre-cast Agarose Gels
0.8% Starter Pak1.2% Starter Pak
2% Starter Pak
4% Starter Pak
9 gels and base9 gels and base
9 gels and base
9 gels and base
G5000-08G5000-01
G5000-02
G5000-04
UltraPure Agarose 100 g
500 g
15510-019
15510-027
UltraPure Agarose 1000 100 g 10975-035
100-bp DNA Ladder 50 g 15628-019
123-bp DNA Ladder 100 g
250 g
15613-011
15613-029
1-Kb Plus DNA Ladder 250 g
1,000 g
10787-018
10787-026
Enzymes for Amplification:
Taq DNA Polymerase, Native 100 units
500 units
1,500 units (3 500 units)
18038-018
18038-042
18038-067
Taq DNA Polymerase, Recombinant 100 units
500 units
1,500 units(3 500 units)
10342-053
10342-020
10342-046
PlatinumTaq DNA Polymerase 100 reactions
250 reactions
500 reactions
5000 reactions
10966-018
10966-026
10966-034
10966-083
PlatinumTaq DNA Polymerase High
Fidelity
100 reactions
500 reactions
5,000 reactions
11304-011
11304-029
11304-102
Platinum
Pfx DNA Polymerase(includes PCRx Enhancer Solution) 100 reactions250 reactions
500 reactions
11708-01311708-021
11708-039
Continued on next page
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22
Related Products, Continued
Product Size Cat. No.
Enzymes for Amplification, continued:
eLONGase Enzyme Mix 100 reactions
500 reactions
10480-010
10480-028PCR SuperMix 100 reactions 10572-014
PCR SuperMix High Fidelity 100 reactions 10790-020
Platinum PCR SuperMix 100 reactions 11306-016
Other Modifying Enzymes:
DNase I, Amplification Grade 100 units 18068-015
RNaseOUT Recombinant 5,000 units 10777-019
Ribonuclease Inhibitor
Ribonuclease H 30 units120 units
18021-01418021-071
SuperScript II Reverse Transcriptase 2,000 units
10,000 units
4 10,000 units
18064-022
18064-014
18064-071
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Purchaser Notification
Limited Use Label
License No: 4:Products for PCR
that include norights to perform
PCR
This product is optimized for use in the Polymerase Chain Reaction (PCR)
covered by patents owned by Roche Molecular Systems, Inc. and F.
Hoffmann-La Roche, Ltd. (Roche). No license under these patents to use
the PCR process is conveyed expressly or by implication to the purchaserby the purchase of this product. A license to use the PCR process for
certain research and development activities accompanies the purchase of
certain reagents from licensed suppliers such as Invitrogen, when used in
conjunction with an Authorized Thermal Cycler, or is available from
Applied Biosystems. Further information on purchasing licenses to practice
the PCR process may be obtained by contacting the Director of Licensing
at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California
94404 or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue,
Alameda, California 94501.
Limited UseLabel License 5:SuperScript
Reverse
Transcriptase
The purchase of this product conveys to the buyer the non-transferable right
to use the purchased amount of the product and components of the product in
research conducted by the buyer (whether the buyer is an academic or for-
profit entity). The buyer cannot sell or otherwise transfer (a) this product (b)
its components or (c) materials made using this product or its components to
a third party or otherwise use this product or its components or materials
made using this product or its components for Commercial Purposes. The
buyer may transfer information or materials made through the use of this
product to a scientific collaborator, provided that such transfer is not for any
Commercial Purpose, and that such collaborator agrees in writing (a) not totransfer such materials to any third party, and (b) to use such transferred
materials and/or information solely for research and not for Commercial
Purposes. Commercial Purposes means any activity by a party for
consideration and may include, but is not limited to: (1) use of the product or
its components in manufacturing; (2) use of the product or its components to
provide a service, information, or data; (3) use of the product or its
components for therapeutic, diagnostic or prophylactic purposes; or (4)
resale of the product or its components, whether or not such product or its
components are resold for use in research. Invitrogen Corporation will not
assert a claim against the buyer of infringement of patents owned by
Invitrogen and claiming this product based upon the manufacture, use or sale
of a therapeutic, clinical diagnostic, vaccine or prophylactic product
developed in research by the buyer in which this product or its components
was employed, provided that neither this product nor any of its components
was used in the manufacture of such product. If the purchaser is not willing
to accept the limitations of this limited use statement, Invitrogen is willing to
accept return of the product with a full refund. For information on
purchasing a license to this product for purposes other than research, contact
Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue,
Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500.
Continued on next page
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Purchaser Notification, Continued
Limited Use
Label LicenseNo: 18:
RNaseOUT
Ribonuclease
Inhibitor
This product is the subject of U.S. Patent No. 5,965,399 owned by
Invitrogen Corporation. The purchase of this product conveys to the buyer
the non-transferable right to use the purchased amount of the product and
components of the product in research conducted by the buyer (whether thebuyer is an academic or for-profit entity). The buyer cannot sell or otherwise
transfer (a) this product (b) its components or (c) materials made using this
product or its components to a third party or otherwise use this product or its
components or materials made using this product or its components for
Commercial Purposes. The buyer may transfer information or materials
made through the use of this product to a scientific collaborator, provided
that such transfer is not for any Commercial Purpose, and that such
collaborator agrees in writing (a) to not transfer such materials to any third
party, and (b) to use such transferred materials and/or information solely for
research and not for Commercial Purposes. Commercial Purposes means any
activity by a party for consideration and may include, but is not limited to:(1) use of the product or its components in manufacturing; (2) use of the
product or its components to provide a service, information, or data; (3) use
of the product or its components for therapeutic, diagnostic or prophylactic
purposes; or (4) resale of the product or its components, whether or not such
product or its components are resold for use in research. Invitrogen
Corporation will not assert a claim against the buyer of infringement of the
above patents based upon the manufacture, use or sale of a therapeutic,
clinical diagnostic, vaccine or prophylactic product developed in research by
the buyer in which this product or its components was employed, provided
that neither this product nor any of its components was used in themanufacture of such product. If the purchaser is not willing to accept the
limitations of this limited use statement, Invitrogen is willing to accept
return of the product with a full refund. For information on purchasing a
license to this product for purposes other than research, contact Licensing
Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad,
California 92008. Phone (760) 603-7200. Fax (760) 602-6500.
OtherTrademarks
TRIzol is a registered trademark of Molecular Research Center, Inc.
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Technical Service
World WideWeb
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Once connected to the Internet, launch your Web browser (InternetExplorer 5.0 or newer or Netscape 4.0 or newer), then enter the
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Contact Us For more information or technical assistance, call, write, fax, oremail. Additional international offices are listed on our Web page
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Corporate Headquarters:
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Carlsbad, CA 92008 USA
Tel: 1 760 603 7200
Tel (Toll Free): 1 800 955
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Fax: 1 760 602 6500
E-mail:
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Invitrogen Japan K.K.
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Bldg. 4F
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E-mail:[email protected]
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Tel: +44 (0) 141 814 6100
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To request an MSDS, visit our Web site at www.invitrogen.com.
On the home page, go to Technical Resources, select MSDS,
and follow instructions on the page.
Continued on next page
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Corporate Headquarters:Invitrogen Corporation1600 Faraday AvenueCarlsbad, California 92008Tel: 1 760 603 7200Tel (Toll Free): 1 800 95 5 6 28 8Fax: 1 760 603 722 9Email: [email protected]
European Headquarters:Invitrogen Ltd
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