CompleteⅡ 1st strand cdna Synthesis Kit

Transcription

1 Instruction Manual CompleteⅡ 1st strand cdna Synthesis Kit Catalog # GM30401, GM30402 Green Mountain Biosystems. LLC Web: Tel: Sales: Sales@ greenmountainbio.com Support: Support@ greenmountainbio.com Address: 755 Watershed Ct., Ann Arbor, MI 48105

2 Components... 3 Storage Conditions... 3 Additional Required Reagents... 3 General Information... 3 Introduction... 3 Quality Control... 4 Choice of Primers for Reverse Transcription:... 4 Detailed Operating Instructions... 5 Protocol... 5 Guidelines for PCR reaction... 5 Guidelines for qpcr reaction... 5 Store cdna at -20 C or used immediately for qpcr reaction FAQs and Troubleshooting... 6 Prevent RNase contamination... 6 Choosing Primers for PCR... 6 Choosing Primers for qpcr... 7 Limited Product Warranty... 7 Green Mountain Biosystems Page2 Version 3.2B 7/2015

3 Components Component GM30401 (50 rxn/20 μl/rxn) GM30402 (100 rxn/20 μl/rxn) RNase free ddh2o 1 ml 1 ml 2 RT Mix a 500 μl 1 ml CompleteⅡ Enzyme Mix b 100 μl 200 μl Oligo (dt)23 VN (50μM) 50 μl 100 μl Random hexamers (50 ng/μl) 50 μl 100 μl a. Contain 1 mm each dntp. b. Contain RNase inhibitor. Storage Conditions Store CompleteⅡ Enzyme Mix and components at -20. Additional Required Reagents Template RNA Primers RNase free microcentrifuge tube of 1.5 ml or 0.2 ml PCR tube Water bath or PCR instrument Ice General Information Introduction Complete II 1 st Strand cdna Synthesis Kit contains Complete II Reverse Transcriptase, a brand new reverse transcriptase based on genetic optimaization of M-MLV (RNase H-) Reverse Transcriptase. Complete II Reverse Transcriptase has a substantial increase in thermal stability over previous generations. Complete II Reverse Transcriptase has a half-life of more than 240 minutes at 50 C and can remain stable for a long time periods at 55 C. Complete II Reverse Transcriptase is ideal for reverse transcription of RNA templates with secondary structures. Furthermore, Complete II Reverse Transcriptase contains point mutations that increase the efficiency of reverse transcription reactions. The enzyme has a higher affinity for template RNA and synthesizes full-length cdna as long as 20 kb, including plant tissue RNA. Complete II 1st strand cdna Synthesis Kit contains all components needed to synthesize high-quality 1st strand cdna. Reaction products can be used in subsequent PCR, qpcr and PCR cloning. 2 RT Mix contains optimized buffers and dntp. Enzyme Mix contains Complete Reverse Transcriptase and RNase inhibitor. Green Mountain Biosystems Page3 Version 3.2B 7/2015

4 Oligo dt18 and Random hexamers can be chosen as primers for reverse transcription when needed. Oligo dt23vn in Kit has stronger anchoring ability for Poly A + RNA, which makes the reverse transcription efficiency higher compared to Oligo dt18. Select Oligo dt23vn, random hexamers or gene specific primers as a reverse transcription primer. Quality Control All of the components are tested to be free of exonuclease, exonuclease or RNase. 1. A clear 20.0 kb DNCH gene band can be detected after agarose gel electrophoresis and EB staining when using 500 ng Hela cell total RNA as template and Oligo dt23 VN as primer for a PCR reaction (45 min at 50 C). 2. A clear 550 bp β- actin gene band can be detected after agarose gel electrophoresis and EB staining when using 100 pg Hela cell total RNA as template and Oligo dt23 VN as primer for a PCR reaction (30 min at 50 C). 3. A clear 4.8 kb (GC-rich) β-actin gene band can be detected after agarose gel electrophoresis and EB staining when using 500 ng Hela cell total RNA as template and Oligo dt23 VN as primer for a PCR reaction(45 min at 55 C) orders of magnitude of the template amount on the numerical value of Ct standard curve, R 2 >0.990, slope between to was determined using by a qrt-pcr performance test taking 1 pg-1 μg Hela cell total RNA as template and Oligo dt23 VN as primer (30 min at 50 C). Choice of Primers for Reverse Transcription: Primers Choosing for PCR 1. Oligo (dt) 23VN is preferred for most applications with eukaryotic mrna as template, because it ensures that all cdna copies terminate at the 3 end of the mrna and produces the longest contiguous cdna. 2. Random hexamers provides random priming sites covering the entire RNA templates including both mrnas, rrna and trna. Random hexamers can be choose while using templates with complex secondary structure or GC-rich, and Oligo (dt) 23VN or gene specific primers (GSP) cannot effectively synthesize cdna,. 3. When a gene-specific primer (GSP) is used in a cdna synthesis reaction, the cdna product can be used only for amplification of that transcript. Primers Choosing for qpcr Mixed Oligo dt23vn with Random hexamers allows cdna applications at each site of mrna with the same rate which improve the repeatability of quantitative research. Green Mountain Biosystems Page4 Version 3.2B 7/2015

5 Detailed Operating Instructions Protocol Guidelines for PCR reaction 1. RNA template denaturation. Set up the following reaction mixture in an RNase free centrifuge tube. Reagent Amount per reaction(μl) 2 RT Mix 10 μl Oligo (dt)23 VN (50 μm) Or Random Primer (50ng/μl) Or Gene Specific Primer (2 μm)) Template RNA Total RAN: 10 pg-5 μg Poly(A) + RNA: 10 pg-500 ng RNase free ddh2o To 8 μl 2. Incubate the reaction at 65 Cfor 5 min then immediate place on ice for 2 min.rna template denaturation will help open the secondary structure improve the yield of 1 st strand cdna. Do not omit this denaturation step for cdna longer than 3 kb. 3. Prepare the 1 st strand cdna synthesis reaction mixture in an RNase free centrifuge tube as follows. Mix gently with a pipette. Component Volume 2 RT Mix 10 μl 5 Complete II Enzyme Mix 2 μl Denatured RNA 8 μl 4. Use the following conditions for the1 st strand cdna synthesis reaction. Temperature Time min min 85 5 min 1. This step is required only when using the Random hexamers; omit if using Oligo dt23vn or Gene Specific Primers. 2. For templates with a complex secondary structure, increase temperature to 55 C. 5. The cdna can be used immediately for PCR reactions, stored at -20 for six months, or at-80 C for long-term storage. Avoid repeated freezing and thawing of cdna. Guidelines for qpcr reaction 1. RNA template denaturation. Set up the following reaction mixture in an RNase free centrifuge tube. Green Mountain Biosystems Page5 Version 3.2B 7/2015

6 Reagent Amount per reaction(μl) 2 RT Mix 10 μl Complete II Enzyme Mix 2μl Oligo (dt)23 VN (50 μm) Random hexamers (50ng/μl) Template RNA Total RAN: 10 pg-1 μg Poly(A)+ RNA: 10 pg-100 ng RNase free ddh2o To 20 μl 2. Use the following conditions for the1 st strand cdna synthesis reaction. Temperature Time min min 85 5 min *For templates with complex secondary structure, increase temperature to 55 C. 3. The cdna can be used immediately for PCR reactions, stored at -20 for six months, or at-80 C for long-term storage. Avoid repeated freezing and thawing of cdna. Store cdna at -20 C or used immediately for qpcr reaction. FAQs and Troubleshooting Prevent RNase contamination 1. Keep the experiment area clean; Wear clean gloves and masks, and use new centrifuge tubes, tips and other supplies to ensure experiment is RNase free. Choosing Primers for PCR 1. Oligo (dt) 23VN hybridizes at high efficiency to the 3 poly (A) region found in most mature eukaryotic mrna. It is the first choice for most cases and generally results in a high yield the full-length cdna. 2. Random hexamers have the lowest specificity. All RNA, including mrna, rrna and trna can be templates of Random hexamers. When the target area of RNA has a complex secondary structure or is GC-rich, and Oligo (dt)18 or gene specific primers (GSP) cannot effectively synthesize cdna, select Random hexamers. 3. Gene specific primers (GSP) have the highest specificity. But under some circumstances, GSP used for PCR reaction cannot effectively synthesize cdna. Select Oligo (dt)18 in this case. Green Mountain Biosystems Page6 Version 3.2B 7/2015

7 Choosing Primers for qpcr 1. Oligo dt23vn mixed with Random hexamers can lead to cdna synthesis at equivalent efficiency and helps to improve the reproducability of quantitative results. Limited Product Warranty Green Mountain Biosystems LLC is committed to providing our customers with high-quality goods and services. Our goal is to ensure that every customer is 100% satisfied with our products and our service. All our products are warranted to perform according to specifications stated on the certificate of analysis. The Company will replace, free of charge, any product that does not meet those specifications. This warranty limits the Company s liability to only the price of the product. No warranty is granted for products beyond their listed expiration date. No warranty is applicable unless all product components are stored in accordance with instructions. The Company reserves the right to select the method(s) used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order. Green Mountain Biosystems LLC shall have no responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever. The above limited warranty is sole and exclusive. No other warranty is made, whether expressed or implied, including any warranty of merchantability or fitness for a particular purpose. All rights reserved. For research use only. Not intended for any animal or human therapeutic or diagnostic use. Green Mountain Biosystems Page7 Version 3.2B 7/2015