cDNA Synthesis System

1 Instruction ManualcDNA Synthesis SystemCAT. NO. 18267-013 Table of Contentsi1. Notices to Customer .. Important Information .. Precautions ..12. Overview .. The cDNA Synthesis System .. cDNA Libraries .. Choosing a Vector System .. mRNA Purification .. cDNA Synthesis : The First Strand Reaction .. cDNA Synthesis : The Second Strand Reaction .. Hairpin-Primed Synthesis .. Okayama and Berg Procedure .. Gubler and Hoffman Procedure .. cDNA Synthesis System : Modification of the Gubler and ..7 Hoffman First Strand Reaction .. Second Strand Reaction .. Cloning Double-Stranded cDNA.

2 Tailing with Terminal Transferase .. Blunt-End Ligation .. Addition of Linkers ..103. Methods .. Components .. mRNA Purification .. General Comments .. First Strand Reaction .. Second Strand Reaction .. Advance Preparations .. First Strand Reaction .. Second Strand Reaction .. First Strand Synthesis .. Second Strand Synthesis .. Protocol 1: cDNA Synthesis for Linker Addition .. Protocol 2: cDNA Synthesis for Tailing .. Analysis of the cDNA Products .. First Strand Yield .. Second Strand Yield .. Amount Recovered after RNase H Digestion .. Gel Analysis.

3 Advance Preparations ..20 Table Procedure ..214. Troubleshooting .. mRNA Purification .. First Strand Reaction .. Second Strand Reaction .. General Guidelines ..245. References ..256. Related Products .. of Procedures for the cDNA Synthesis System .. cDNA Synthesis .. Strand Synthesis .. of Second Strand Procedures Using the cDNA Synthesis System .. Flow Diagram ..13DH5 , DH5 FT ,DH10B , ELECTROMAX , GENETRAPPER ,MESSAGEMAKER , FOCUS , MAX EFFICIENCY ,SUPERSCRIPT , TECH-LINESM, and ULTRAMAX are marks of Invitrogen is a trademark of Molecular Bio-Products, is a registered trademark of Molecular Research Center, to Important InformationThis product is authorized for laboratory research use only.

4 The product has notbeen qualified or found safe and effective for any human or animal diagnostic ortherapeutic application. Uses for other than the labeled intended use may be aviolation of applicable PrecautionsWarning: This product contains hazardous reagents. It is the end-user sresponsibility to consult the applicable MSDS(s) before using this product. Disposalof waste organics, acids, bases, and radioactive materials must comply with allappropriate federal, state, and local regulations. If you have any questionsconcerning the hazards associated with this product, please call the InvitrogenEnvironmental Health and Safety Chemical Emergency hotline at (301) cDNA Synthesis SystemThe cDNA Synthesis System provides the materials needed to rapidly and reliablysynthesize double-stranded cDNA from mRNA.

5 It uses cloned Moloney MurineLeukemia Virus (M-MLV) reverse transcriptase to synthesize first strand cDNA froman mRNA population. Figure 1 is a summary of the two procedures that may beused to prepare double-stranded cDNA for tailing or linker addition. The one-tubesystem allows second strand cDNA to be synthesized immediately after the firststrand reaction, thereby improving the yield of double-stranded cDNA by eliminatingextraction and precipitation between steps. Furthermore, the double-stranded cDNAcan be cloned into either plasmid or bacteriophage M-MLVR everse TranscriptaseProcedure IIDNA Polymerase IDNA LigaseSecond Strand cDNAS econd Strand cDNAds cDNA foruse with linkersFirst Strand cDNAds cDNA forhomopolymer tailingProcedure IDNA Polymerase IDNA LigaseRNase HRNase HFigure 1.

6 Outline of procedures for the cDNA Synthesis LibrariesBacteria or bacteriophage containing cDNA copies of an mRNA populationconstitute a cDNA library. A good library should be large enough to containrepresentatives of all sequences of interest, and the cDNA inserts should be full-length copies of the original mRNA. Sequences at both the 5 and 3 ends ofmRNAs contain valuable information needed for full understanding of geneexpression and of a cDNA library from mRNAs is an essential step for many studies inmolecular biology. A cDNA clone isolated from a cDNA library provides a means ofestablishing the fully processed form of a message initially transcribed from agenomic sequence containing intervening sequences (introns).

7 It also provides aprobe for examining the structure of the gene, determining its location in thegenome, or developing in vitroor recombinant expression systems. Comparing twolibraries constructed from different tissues or different stages of a developmentalsystem can reveal developmentally regulated genes. For these and other types ofstudies, a high quality library is procedures for synthesizing cDNA have been developed, most attempting tomaximize the amount of cDNA produced from a limited amount of mRNA. In most ofthese procedures, the completeness of the cDNA Synthesis is variable andunpredictable.

8 This variation can enter in any of the three steps of cDNA Synthesis :mRNA isolation, first strand cDNA Synthesis , and second strand cDNA quality of the mRNA preparation, the purity of the enzyme and reagents used inthe first strand reaction, and the choice of second strand method affect the quantityof sequence information retained in the a Vector SystemTwo types of vectors are in general use for cDNA cloning in E. coli:plasmids andbacteriophage lambda derivatives. A number of factors affect the choice of vector,which in turn affects the method of cDNA Synthesis .

9 In choosing a vector, considerthe many clones are needed to provide a reasonable chance of findingthe gene of interest? critical is it to have a complete copy of the mRNA containing the 5 end of the message? important is it to obtain a clone that can express a protein product? familiar and comfortable are you with the manipulation andpropagation of plasmids or phage?The number of clones needed to make a library complete depends upon the relativeabundance of the target mRNA in the total mRNA pool (1). Therefore, it is advan-tageous to use an enriched source of the target mRNA if possible.

10 The enrichmentcan be biological (as in the case of a developmentally regulated gene) or physical(hybrid selection or immunoprecipitation). The size of a library needed to obtain anmRNA as a cDNA clone generally requires 104to 105recombinants (1). Eitherplasmids or phage vectors can yield this number of clones; however, the efficiencyof cloning with phage vectors is generally higher and requires less mRNA method used to synthesize the double-stranded cDNAs will influence theamount of sequence information at the 5 end of the mRNAs that is preserved in thecDNA library (2).