By Juris Steinbergs, Aleksander Tsimanis (auth.), Professor Dr. Valdis Berzins (eds.)
This guide introduces the reader to bacic tools utilized in the isolation, cloning and research of genetic fabric. The protocols comprise RT-PCR amplification, gene cloning, hybridization research and sequencing of nucleic acids, PCR-based site-specific mutagenesis, research of protein DNA-specific interplay, cell-free protein synthesis and product electrophoretic and immunological research. every one protocol comprises brief history info, an in depth description of the required fabrics, step by step tactics, a troubleshooting advisor and important functional hints.
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Additional resources for Basic Cloning Procedures
Note. Reading sequences close to the primer. The general conditions described in this manual should be followed for sequencing from the primer up to 300-400 nucleotides. If one is interested only in the sequences close to the primer «200 nucleotides), it is possible to dilute the labeling mixture further (make a 1:20 dilution of the stock reagent) and keep both reaction times to 3-5 min. The gel should be run only until the first blue dye has migrated about 80 % of the length of the gel. When reading sequences within 20 nucleotides of the 3' end of primer, it is essential that a sufficient amount of the template DNA and primer be present.
2). Most of inserted genes can be obtained as full-size sense or antisense RNA transcripts and the yields can reach several hundred transcripts per molecule of template DNA. The use of synthetic Mes r"lt~IO";"g ,'e) _-----'»~ Part '-------' 1 T7 promoter of transcription vector SP6 promoter Restriction digest of MCS and insertion of gene, ligation and cloning ----1,------,VIIIIIIIII T7 promoter L '+'R A 3' 111111111111111 RNA 5' R SP6 promote~estriction of ~ / IIIIIIIIII ~ 1 T7 -~ I recombinant DNA VIIIIIIIIII Transcription Fig.
83 M boric acid Buffers 32 E. 5 mmolll dCTP 80 80 80 80 2 mmolll ddGTP 2 2 mmolll ddATP 2 2 mmolll ddTTP 2 2 2 mmolll ddCTP 5M NaCl 5 5 5 5 173 173 173 173 Redist. water 500 500 Final volume 500 500 Procedure Template DNA Denaturation Note. This first step is necessary only when double-stranded templates are used. 1. 5 ml microcentrifuge tube. 2. 5 M NaOH. Let stand for 5 min at room temperature. 3. l of 5 M ammonium acetate. Mix well. 4. l of ethanol. 5. Spin the tubes in a micro centrifuge for 15 min.