AMV LongAmp Taq RT-PCR Kit

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1 DNA AMPLIFICATION & PCR AMV LongAmp Taq RT-PCR Kit Instruction Manual NEB #E5300S Store at 20 C

2 ISO 9001 Registered Quality Management ISO Registered Environmental Management ISO Registered Medical Devices PROTOSCRIPT, LONGAMP and NEW ENGLAND BIOLABS are registered trademarks of New England Biolabs, Inc. PRIMERSELECT is a trademark of DNASTAR Inc. icycler is a registered trademark of Bio-Rad Laboratories, Inc. Purchase of this product provides the purchaser with a non-exclusive license to use ProtoScript AMV LongAmp Taq RT-PCR Kit for research purposes only. This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

AMV LongAmpTaq RT-PCR Kit Table of Contents: Introduction...2 Quality Controls....2 First Strand cdna Synthesis Protocols...2 PCR Amplification...3 General Information for Successful cdna Synthesis.

3 AMV LongAmpTaq RT-PCR Kit Table of Contents: Introduction...2 Quality Controls....2 First Strand cdna Synthesis Protocols...2 PCR Amplification...3 General Information for Successful cdna Synthesis...4 Template RNA...4 First Strand cdna Synthesis Reaction...4 Choice of Primers for Reverse Transcription...4 PCR Primers...5 PCR Amplification...5 Troubleshooting Guide...6 References...7 Appendix....7 Ordering Information....8 Kit Components: All kit components should be stored for one year at 20 C except where noted. Stability can be extended by storing at 80 C. Keep d(t) 23 VN and Random Primer Mix at 20 C AMV Enzyme Mix (10X) µl AMV Reaction Mix (2X) µl Oligo d(t) 23 VN* (50 µm)**...70 µl Random Primer Mix (60 µm)** µl LongAmp Taq 2X Master Mix ml Nuclease-free H 2 O...1 ml *V = A,G or C; N = A, G, C or T **Contains 1 mm dntp 1

4 Introduction: The AMV LongAmp Taq RT-PCR Kit combines two powerful mixes, AMV Enzyme Mix and LongAmp Taq 2X Master Mix for 2-step RT-PCR applications. The two mixes require minimal handling during reaction setup and yet offer consistent and robust RT-PCR reactions. AMV Enzyme Mix can carry out first strand cdna synthesis reactions with a broader optimal reaction temperature from 37 C to 50 C. LongAmp Taq 2X Master Mix offers consistent and robust amplification from more than 14 kb cdna products. Primer d(t) 23 VN is included for anchored priming at the polya-tail region of messenger RNAs. Random Primer Mix is an optimized mix of hexamer and d(t) 23 VN primers, offering good coverage of RNA templates. Quality Controls: The performance of AMV LongAmp Taq RT-PCR Kit is tested in an RT reaction using human Jurkat total RNA with primer d(t) 23 VN. The sensitivity of the kit is verified by the detection of GAPDH transcript in 20 pg total RNA after 35 cycles. The length of cdna achieved is verified by the detection of a 5.5 kb amplicon of the p532 gene. First Strand cdna Synthesis Protocols Thaw system components and put on ice. A control reaction without reverse transcriptase is recommended to examine the DNA contamination in the samples. 1. Mix RNA sample and primer d(t) 23 VN in two sterile RNase-free microfuge tubes. Total RNA d(t) 23 VN (50 μm) nuclease-free H 2 O Total Volume 1 6 μl (10 pg 1 μg) 2 μl variable 8 μl 2. Denature RNA for 5 minutes at 70 C. Spin briefly and put promptly on ice. This step is optional. However, it improves the cdna yield for long messenger RNAs and GC-rich RNA regions. 3. Add the following components to one tube. AMV Reaction Mix 10 µl AMV Enzyme Mix 2 µl To the negative control tube, add the following: AMV Reaction Mix 10 µl H 2 O 2 µl 2

5 4. Incubate the 20 μl cdna synthesis reaction at 42 C for one hour. If Random Primer Mix is used, an incubation step at 25 C for 5 min is recommended before the 42 C incubation. 5. Inactivate the enzyme at 80 C for 5 minutes. Dilute reaction to 50 µl with 30 µl H 2 O for PCR. The cdna product should be stored at 20 C. For downsteam PCR amplification, the volume of cdna product should not exceed 1/10 of the PCR reaction volume. PCR Amplification: We recommend 2 5 µl of the diluted cdna product for a 50 µl PCR reaction. Mix the LongAmp Taq 2X Master Mix by inverting before use. 1. Mix the following components in a PCR tube on ice: LongAmp Taq 2X Master Mix 25 μl 10 µm forward primer 1 μl 10 µm reverse primer 1 μl Diluted cdna 2 5 μl H 2 O variable Total Volume 50 μl 2. Mix gently. Overlay with mineral oil if the thermal cycler lacks a heated lid. 3. The following PCR cycling conditions are recommended for 0.2 ml thinwall PCR tubes on Bio-Rad icycler or similar thermocyclers. For other PCR tubes or cyclers, it may be necessary to modify the cycle times. INITIAL DENATURATION 95 C 30 SECONDS Cycles 94 C C 68 C seconds 30 seconds 50 seconds per kb Final Extension 68 C 5 minutes 2-temperature PCR protocol (When using primers with annealing temperature above 60 C). INITIAL DENATURATION 95 C 30 SECONDS Cycles 94 C C seconds 60 seconds per kb Final Extension C 5 minutes Final Soak 4 10 C 4. Analyze 5 μl of PCR products by agarose gel electrophoresis. 3

6 General Information for Successful cdna Synthesis: Template RNA Intact RNA of high purity is essential for sensitive RT-PCR detection. RNA should have a minimum A 260 /A 280 ratio of 1.7 or higher. Either total RNA or mrna can be used in the reverse transcription reaction. Total RNA is generally sufficient for most RT-PCR analyses. However, if desired mrna can be easily obtained using a PolyA Spin mrna Isolation Kit (NEB #S1560) or Magnetic mrna Isolation Kit (NEB #S1550). The amount of RNA required for detection depends on the abundance of the transcript of interest. In general 1 ng to 1 μg total RNA or ng mrna are recommended. First Strand cdna Synthesis Reaction: Denaturation of RNA and primer at 70 C for 5 minutes can remove secondary structures that may impede long cdna synthesis. However, this step can be omitted in some cases (unpublished results). We recommend incubation at 42 C for one hour for maximum yield and length. However, many targets can be detected after a much shorter incubation time. For example, 5 minutes incubation is enough for a 2 kb cdna synthesis. Higher reaction temperature up to 60 C can be used for difficult targets of high secondary structures. Choice of Primers for Reverse Transcription: Oligo d(t) priming is preferred for most applications because it ensures that all cdna copies terminate at the 3 end of the mrna and produces the longest contiguous cdna. An anchored oligo-d(t) primer [d(t) 23 VN] forces the primer to anneal to the start of the polya tail, thereby preventing priming at internal sites in the polya tail (1). However, two other priming choices are possible if desired. The Random Primer Mix is an optimized mix of hexamer and d(t) 23 VN primers. It provides random priming sites covering the entire RNA templates including both mrnas and non-polyadenylated RNAs (such as ribosomal RNAs). The Random Primer Mix yields shorter cdnas on average and can be used for the detection of multiple short RT-PCR products. Random Primer Mix offers good performance in a wide range of RNA templates. When a gene-specific primer is used in a cdna synthesis reaction, the cdna product can be used only for amplification of that transcript. This priming method gives good results when the amount of RNA is limiting (below 10 ng) and only one particular cdna is desired. 4

7 Recommended primer concentration: PRIMER OLIGO d(t) 23 VN RANDOM PRIMER MIX SPECIFIC PRIMER Final conc. 5 μm 6 μm μm PCR Primers: Specific primers for PCR should be designed with the aid of a primer design computer program to achieve best results, such as PrimerSelect (DNAStar Inc, Madison, MI) and Primer3 ( To minimize the complication introduced by contaminating genomic DNA, use primers that span an exon-exon boundary of the mrna. PCR Amplification: Most targets can be efficiently amplified using 1/10 (2 μl out of 20 μl cdna synthesis reaction) or much less of the cdna product (2). A final concentration of 0.2 μm for each primer is recommended for PCR; however, it can vary between 0.05 μm to 1 μm. The extension step using LongAmp Taq 2X Master Mix is recommended at C with an extension rate of 50 seconds per kb. For GC rich targets, we recommend to include DMSO up to 10%. Use of thin-wall 0.2 ml PCR tubes and a manual hot-start may increase the PCR sensitivity and yield. A manual hot-start is done by assembling reactions into tubes placed on ice. The reaction tubes are transferred to a PCR machine with a block preheated at 95 C. Upon placement of the tubes, the cycler is immediately started. 5

8 Troubleshooting Guide: Problem Suggestion Low Yield of cdna Check the integrity of the RNA by denaturing agarose gel electrophoresis (2). RNA should have a minimum A 260 /A 280 ratio of 1.7 or higher. Ethanol precipitation followed by a 70% ethanol wash can remove most contaminants such as EDTA and guanidinium. Precipitation with lithium chloride can remove polysaccharides (2). Phenol/chloroform extraction and ethanol extraction can remove contaminant proteins such as proteases (2). Some target RNA may contain strong pauses for RT; Use random priming instead of d(t) 23 VN. Use sufficient amount of RNA. Low Yield of PCR Product Products of Wrong Size Check the primer design using computer software. Optimize the annealing temperature in a 1 2 C step. A primer concentration of 0.2 μm is satisfactory for most PCR reactions. However, sensitivity and yield of RT-PCR reactions can be improved by increasing the primer concentration to above 0.5 μm. Lower primer concentration between 0.07 μm to 0.2 μm may improve specificity. Increase cycling numbers up to 45 cycles. Do a manual hot-start. Use thin-wall 0.2 ml PCR tubes. Try a touch-down PCR protocol (4). Always do a negative control reaction in the absence of reverse transcriptase. In cases where the RNA sample is contaminated with genomic DNA, treat with DNase I before cdna synthesis (5). Design primers spanning an exon-exon boundary. 6

9 References: 1. Liao, J. and Gong, Z. (1997) Biotechniques 23, Van Gilst, M.R. et al. (2005) PLoS Biology 3, Sambrook, J. and Russel, D.W. (2001). Molecular Cloning: A Laboratory Manual, (3rd ed.), (pp and ). Cold Spring Harbor: Cold Spring Harbor Laboratory Press. 4. Don, R.H. et al. (1991) Nucleic Acid Research 19, Aguila et al. (2005) BMC Molecular Biology 6, 9. Appendix: Supplied Components: 1X AMV Enzyme Mix 0.1 unit/µl AMV Reverse Transcriptase 1 unit/µl Murine RNase Inhibitor 1X AMV Reaction Mix 50 mm Tris-Acetate (ph 8.4) 75 mm KOAc 10 mm Mg(OAc) mm DTT 1 mm dntps each 7

10 Ordering Information PRODUCT NEB # SIZE AMV LongAmp Taq RT-PCR Kit E5300S 30 reactions KIT COMPONENTS SOLD SEPARATELY AMV Reverse Transcriptase M0277S/L 200/1,000 units Murine RNase Inhibitor M0314S/L 3,000/15,000 units AMV First Strand cdna Synthesis Kit E6550S 30 reactions Random Primer Mix S1330S 1.0 A 260 units LongAmp Taq 2X Master Mix M0287S/L 100/500 reactions COMPANION PRODUCTS ssrna Ladder N0362S 25 gel lanes 1 kb DNA Ladder N3232S/L 200/1,000 gel lanes 100 bp DNA Ladder N3231S/L 100/500 gel lanes ProtoScript Taq RT-PCR Kit E6400S 30 reactions Deoxynucleotide Solution Mix N0447S/L 8/40 µmol of each polya Spin mrna Isolation Kit S1560S 8 isolations LongAmp Taq DNA Polymerase M0323S/L 500/2,500 units LongAmp Taq PCR Kit E5200S 100 reactions 8

12 DNA CLONING DNA AMPLIFICATION & PCR EPIGENETICS RNA ANALYSIS LIBRARY PREP FOR NEXT GEN SEQUENCING PROTEIN EXPRESSION & ANALYSIS CELLULAR ANALYSIS USA New England Biolabs, Inc. 240 County Road Ipswich, MA Telephone: (978) Toll Free: (USA Orders) Toll Free: (USA Tech) Fax: (978) CANADA New England Biolabs, Ltd. Telephone: (905) Toll Free: Fax: (905) Fax Toll Free: CHINA, PEOPLE S REPUBLIC New England Biolabs (Beijing), Ltd. Telephone: / Fax: info@neb-china.com FRANCE New England Biolabs France Free Call: Free Fax: info.fr@neb.com GERMANY & AUSTRIA New England Biolabs GmbH Telephone: +49/(0)69/ Free Call: 0800/ (Germany) Free Call: 00800/ (Austria) Fax: +49/(0)69/ Free Fax: 0800/ (Germany) info.de@neb.com JAPAN New England Biolabs Japan, Inc. Telephone: +81 (0) Fax: +81 (0) info@neb-japan.com SINGAPORE New England Biolabs Pte. Ltd. Telephone: Fax: sales.sg@neb.com UNITED KINGDOM New England Biolabs (UK) Ltd. Telephone: (01462) Call Free: Fax: (01462) Fax Free: info.uk@neb.com Version /14

AMV First Strand cdna Synthesis Kit

DNA AMPLIFICATION & PCR AMV First Strand cdna Synthesis Kit Instruction Manual NEB #E6550S Store at 20 C ISO 9001 Registered Quality Management ISO 14001 Registered Environmental Management ISO 13485 Registered