ThermoScript ™ RT-PCR System
Catalog nos. (25 reactions): Catalog nos. (100 reactions): 11146-024
11146-016
11146-057 (w/ Platinum ® Taq DNA polymera) 11146-032 (w/ Platinum ® Taq DNA polymera)
11146-040 (w/ Platinum ® Taq DNA polymera High Fidelity)
Store at -20°C (stability can be extended by storing at -70°C)
Description
The ThermoScript ™ RT-PCR System is designed for the nsitive and reproducible detection and analysis of RNA molecules in a two-step process. ThermoScript ™ RT, an avian rever transcripta with reduced RNa H activity, is engineered to have higher thermal stability,
produce higher yields of cDNA, and produce more full-length cDNA transcripts than AMV RT. cDNA synthesis is performed in the first step using either total RNA or poly(A)+-lected RNA primed with olig
o(dT), random primers or a gene-specific primer, at 50-65°C. In the cond step, PCR is performed in a parate tube using primers specific for the gene of interest. RNA targets from 100 bp to >12 kb can be detected with this system, using 10 pg to 5 Yg of total RNA. PCR is carried out with Platinum ® Taq DNA Polymera or Platinum ® Taq DNA Polymera High Fidelity. Platinum ® Taq DNA Polymera High Fidelity is suitable for templates from 100 bp to >12 kb. Platinum ® Taq DNA polymera (1) provides automatic hot-start conditions for incread specificity up to 3 kb.
Reagents are provided for 25 or 100 cDNA synthesis reactions of 20 μl each and 25 or 100 amplification reactions of 50 μl each.
Component 25 rxn kit 100 rxn kit ThermoScript ™ RT (15 U/Yl) 25 μl 100 μl 5X cDNA Synthesis Buffer* 500 μl 500 μl 0.1 M DTT 250 μl 250 μl 10 mM dNTP Mix 100 μl 2 × 250 μl
RNaOUT ™ (40 U/Yl) 25 μl 100 μl
Oligo (dT)20 (50 YM) 25 μl 100 μl
Random Hexamers (50 ng/Yl) 50 μl 250 μl
DEPC-Treated Water 1.25 ml 1.25 ml
E . coli RNa H (2 U/Yl) 50 μl 2 × 50 μl
*250 mM Tris acetate (pH 8.4), 375 mM potassium acetate, 40 mM
magnesium acetate, stabilizer
Catalog numbers 11146-057 (25 rxns) and 11146-032 (100 rxns) include the following, in addition to the components to the left:
Component 25 rxn kit 100 rxn kit
Platinum ® Taq DNA polymera (5 U/Yl) 100 units 250 units
10X PCR buffer Minus Mg 1.0 ml 1.0 ml 50 mM MgCl 2 1.0 ml 1.0 ml Catalog number 11146-040 (100 rxns) includes the following, in addition to the components to the left: Component 100 rxn kit Platinum ®
Taq DNA Polymera High Fidelity (5 U/μl) 100 units 10X High Fidelity PCR Buffer 1.0 ml 50 mM MgSO 4 1.0 ml Quality Control
杨宇宁The Certificate of Analysis (CofA) provides detailed quality control information for each product. The CofA is available on our website at
/cofa, and is archable by product lot number, which is printed on each box .
Summary of Procedure
Part no. 11146.pps MAN0000941 Rev. date: 11 Jun 2010
20 GSP
r andom hexame r C, 30-60 min 50-65°C, 30-60 min 25°C, 10 min ↓ 50-60°C, 20-50 min
↓
85°C, 5 min
↓ Add 1 μl RNa H 37°C, 20 min ↓ Remove 2 μl aliquot for PCR
Water 1 *If less than 1 ng of RNA is ud, reduce the amount of ThermoScript RT in the reaction to 0.5 μl. For technical support, email tech_ For country-specific contact infor
mation, visit .
Page 2 of 4 Important Parameters to Consider
RNA
•High quality intact RNA is esntial for successful full-length cDNA synthesis and successful long RT-
PCR.
•RNA should be devoid of any RNa contamination and aptic conditions should be maintained. •Recommended methods of total RNA isolation include the Micro-to-Midi Total RNA Purification
System (Catalog no. 12183-018) and TRIzol® Reagent
(Catalog no. 15596-026) (2, 3). Oligo(dT)-lection for
poly(A)+ RNA is typically not necessary, although
incorporating this step may improve the yield of
specific cDNAs.
cDNA Synthesis Primers
•Oligo(dT)20 (50 pmoles/reaction) is recommended for priming polyadenylated RNA. U of Oligo(dT)20
allows the detection of multiple transcripts from a
single first-strand reaction.
•Random hexamers (50-250 ng/reaction) are efficient primers for the detection of multiple short RT-PCR
targets. U of more than 50-100 ng primer/Yg of
我会说RNA can increa the yield of short products but may inhibit detection of long targets (>3kb) or rare
transcripts. If random hexamers are ud, the first-
strand reaction must be incubated at 25°C for 10 min
痛苦与快乐
to extend the primers prior to increasing the reaction
temperature for synthesis.
•Gene-specific primers (GSP) should be ud at 10 to
20 pmol/reaction. Specificity of priming may be
improved by optimizing annealing/reaction
temperature.
•Treatment of cDNA with RNa H to remove the complementary RNA prior to PCR is optional. RNa
H digestion will improve the RT-PCR signal of many
targets and is required for the efficient and consistent
amplification of long RT-PCR templates.
cDNA Synthesis Reaction
开创长吉体诗歌的是谁•Denaturation of the RNA template and primer by incubating at 65°C for 5 min is optional. Most targets
can be rever transcribed efficiently without this step.
However, heating the RNA in the abnce of reaction谢谢您老师
buffer and enzyme prior to cDNA synthesis can
remove condary structure that may impede full-
length cDNA synthesis.
•ThermoScript™ RT can be ud at 50-65°C. We recommend incubation at 50-55°C for most RT-PCR
targets. However, incubation at 50-60°C for oligo(dT)
and 50-65°C for gene-specific primers can be
美术的英文employed to reduce condary structure or to improve specificity.
•Most targets can be amplified after only a 30-min incubation for the first-strand reaction. Rare RNAs,
long transcripts, or targets at the 5′ end of long
transcripts benefit from longer incubation times (50-60 min).PCR Primers
• A final primer concentration of 0.2 - 0.4 μM for each primer is generally optimal; however, a primer
titration is recommended for best results.
•Design primers that anneal to quence in exons on both sides of an intron or exon/exon boundary of the mRNA to allow differentiation between amplification of cDNA and potential contaminating genomic DNA. •Primers should not be lf-complementary or complementary to each other at the 3′ ends.
PCR Reactions
•Most targets will be efficiently amplified using 2 Yl or less of the cDNA synthesis reaction.
•The optimum magnesium concentration varies from
1.5 to 3 mM. Generally, 1.82 mM magnesium chloride
for Platinum® Taq DNA polymera and 2.32 mM
magnesium sulfate for Platinum® Taq DNA
Polymera High Fidelity is effective for most primer ts. However, titration of the magnesium
concentration is recommended for the best result.
Each Yl of the cDNA synthesis reaction adds 0.16 mM to the final magnesium concentration in a 50-μl PCR
reaction.
•Asmble the PCR reactions on ice, transfer them to a pre-heated thermal cycler (85-95°C) and immediately start the PCR amplification program.
•The annealing temperature should be 10°C below the melting temperature of the primers ud.
•The optimum extension temperature for Platinum®Taq DNA Polymera High Fidelity is 68°C. The
extension time varies with the size of the amplicon
(approximately 1 min per 1 kb of amplicon).
Page 3 of 4
cDNA Synthesis
1. In a 0.2- or 0.5-ml tube, combine primer (50 μM Oligo(dT)20,
50 ng/μl random primer or 10 μM gene-specific primer), RNA, and dNTP mix and adjust volume to 12 Yl with
DEPC-treated water.
Component Amount
Primer 1 Yl
RNA (10 pg -5 Yg) x Yl
10 mM dNTP Mix 2 Yl
DEPC-treated water to 12 Yl
2. Denature RNA and primer by incubating at 65°C for 5 min
and then place on ice (optional).
3. Vortex the 5X cDNA Synthesis Buffer for 5 s just prior to
u.
4. Prepare a master reaction mix on ice and vortex gently.
Component 1 Reaction 10 Reactions
5x cDNA Synthesis Buffer 4 Yl 40 Yl
0.1 M DTT 1 Yl 10 Yl
苹果手机定时关机RNaOUT™ (40 U/Yl) 1 Yl 10 Yl
DEPC-treated water 1 Yl 10 Yl
ThermoScript™ RT (15 units/Yl)1 Yl* 10 Yl*
*NOTE: If less than 1 ng of template RNA is ud, reduce the amount of ThermoScript™ RT in the reaction to 0.5 μl/reaction
(5 μl/10 reactions). Increa the amount of DEPC-treated
water in the master reaction mix to 1.5 μl/reaction (15 μl/10 reactions).
5. Pipet 8 Yl of master reaction mix into each reaction tube on ice.
6. Transfer the sample to a thermal cycler preheated to the appropriate cDNA synthesis temperature and incubate as follows.
Oligo(dT)20 primed: 30-60 min at 50°C (or 50-60°C)
Gene-specific primed 30-60 min at 50°C (or 50-65°C) Random-hexamer primed: 25°C for 10 min, followed by 20-
50 min at 50°C (or 50-65°C)
7. Terminate the reaction by incubating at 85°C for 5 min.
8. Add 1 Yl of RNa H and incubate at 37°C for 20 min (optional).
9. cDNA synthesis reactions can be stored at -20°C or ud for PCR immediately.
PCR with Platinum®Taq DNA Polymera High Fidelity
U only 10% of the cDNA synthesis reaction (2 Yl) for PCR.
U of 2 μl of 50 mM MgSO4 and 2 Yl of cDNA (0.32 mM magnesium in a 50-Yl PCR) results in a final concentration of
2.32 mM magnesium, which is effective for most primer ts. However, titration of the magnesium concentration with the provided 50 mM MgSO4 is recommended for best results.
1. Add the following to a 0.2- or 0.5-ml, thin-walled, PCR tube: Component 1 Reaction 10 Reactions
10X High Fidelity PCR Buffer 5 Yl 50 Yl
50 mM MgSO4 2
μl 20
μl
10 mM dNTP Mix 1 Yl 10 Yl
10 YM n primer 1 Yl 10 Yl
10 YM antin primer 1 Yl 10 Yl
Platinum®Taq High Fidelity 0.2 Yl 2 Yl
cDNA (from cDNA synthesis reaction) 2 Yl 20 Yl
DEPC-treated water 37.8 Yl 378 Yl
Final volume 50 Yl 500 μl 2. Mix gently and overlay with silicone oil or mineral oil if the
thermal cycler lacks a heated lid.
3. Incubate at 94°C for 2 min, then perform 20 to 40 cycles of
PCR with optimized conditions for your sample (1 min/kb extension time at 68°C).
4. Analyze 10 Yl of the amplified sample by agaro gel
electrophoresis.
PCR with Platinum® Taq DNA Polymera
U only 10% of the cDNA synthesis reaction (2 Yl) for PCR. U of 50 mM MgCl2 and 2 Yl of cDNA will result in a final magnesium concentration of 1.82 mM, which is adequate for most primers and targets. However, titration of magnesium concentration is recommended for best results.
1. Add the following to a 0.2- or 0.5-ml, thin-walled, PCR tube: Component 1 Reaction 10 Reactions 10X PCR Buffer Minus Mg 5 Yl 50 Yl
50 mM MgCl2 1.5 Yl 15 Yl
10 mM dNTP Mix 1 Yl 10 Yl
10 YM n primer 1 Yl 10 Yl
10 YM antin primer 1 Yl 10 Yl
Platinum®Taq DNA polymera 0.4 Yl 4 Yl
(5 U /Yl)
cDNA (from cDNA synthesis reaction) 2 Yl 20 Yl DEPC-treated water 38.1 Yl 381 Yl Final volume 50 Yl 500 μl 2. Mix gently and overlay with silicone oil or mineral oil if the
thermal cycler lacks a heated lid.
3. Incubate at 94°C for 2 min, then perform 20 to 40 cycles of
PCR with optimized conditions for your sample (1 min/kb extension time at 68-72°C)
4. Analyze 10 Yl of the amplified sample by agaro gel
electrophoresis.
Control Reactions
An RT-PCR Primer and Control Set is available parately for monitoring the performance of the system (Cat. Number 10929-016).
1. U 1 ng of the Control RNA in the cDNA Synthesis
Reaction.
2. Perform the PCR using Platinum® Taq DNA Polymera, as described above.
Page 4 of 4 Troubleshooting Guide
Problem Possible cau Possible solution
No RT-PCR product No cDNA synthesis (temperature too high) For the cDNA synthesis step, incubate at 45-50°C.
Incomplete synthesis of target cDNA (condary structure of RNA blocks synthesis) For the cDNA synthesis step, incubate at 50-70°C. For long mRNAs, increa cDNA synthesis incubation time (up to 50 min)
RNa contamination Maintain aptic conditions; add RNaOUT™ (RNa
inhibitor).
Concentration of template RNA in reaction is too low Increa the concentration of template RNA; u 1-5 μg of total RNA or reduce the volume of ThermoScript™ RT ud in the reaction.
RNA has been damaged or degraded Replace RNA.
RT inhibitors are prent in RNA Remove inhibitors in the RNA preparation by an
additional 70% ethanol wash after ethanol
precipation.
Note: Inhibitors of RT include SDS, EDTA,
guanidinium chloride, formamide, sodium
phosphate and spermidine (4).
Cycle number is too low Increa cycle number.
Low yield/low specificity in PCR Reaction conditions not optimal Optimize magnesium concentration.
Optimize the primer concentration
Optimize the annealing temperature and extension
time.
Increa temperature of RT reaction to 50-60°C.
Unexpected bands after electrophoresis RNA contamination with genomic DNA Pre-treat RNA with DNa I.
Redesign PCR primers to anneal to quence in exons
on both sides of an intron in the target gene.
References
1.Westfall, B., Sitaraman, K., Solus, J., Hughes, J., and Rashtchian, A. (1997) Focus®19, 46.
2.Chomczynski, P and Sacchi, N. (1987) Anal. Biochem. 162, 156.
3.Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979) Biochemistry 18, 529
4.
4.Gerard, G.F (1994). Focus®16, 102.
Limited U Label Licen No. 1: Thermostable Polymeras (applies to 11146-057, 11146-032, and 11146-040)
U of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,789,224, 5,618,711, and 6,127,155. The purcha of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchar’s own internal rearch. No right under any other patent claim, no right to perform any patented method, and no right to perform commercial rvices of any kind, including without limitation reporting the results of purchar's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for rearch u only. Diagnostic us under Roche patents require a parate licen from Roche. Further information on purchasing
licens may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
Limited U Label Licen No. 4: Products for PCR that include no rights to perform PCR (applies to 11146-016 and 11146-024)
This product is compatible for u in the Polymera Chain Reaction (PCR) process claimed in patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. No licen under the patents is conveyed expressly, by implication, by estoppel or otherwi to the purchar by the purcha of this product. A licen to u the patented process for certain rearch and develop
ment activities accompanies the purcha of certain reagents of Applied Biosystems and other licend suppliers when ud in conjunction with an authorized thermal cycler, or is available from Applied Biosystems. Further information on purchasing licens may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
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清明节诗句
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Licend to Life Technologies Corporation, under U.S. Patent Nos. 5,338,671; 5,587,287; and foreign equivalents for u in rearch only
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