LEADER 04974nam 2200625Ia 450 001 9910784526803321 005 20200520144314.0 010 $a1-281-01889-9 010 $a9786611018894 010 $a0-08-053682-4 035 $a(CKB)1000000000384330 035 $a(EBL)307130 035 $a(OCoLC)173520343 035 $a(SSID)ssj0000204811 035 $a(PQKBManifestationID)11172539 035 $a(PQKBTitleCode)TC0000204811 035 $a(PQKBWorkID)10189179 035 $a(PQKB)11159731 035 $a(Au-PeEL)EBL307130 035 $a(CaPaEBR)ebr10186623 035 $a(CaONFJC)MIL101889 035 $a(MiAaPQ)EBC307130 035 $a(PPN)182573478 035 $a(EXLCZ)991000000000384330 100 $a19980814d1998 uy 0 101 0 $aeng 135 $aur|n|---||||| 181 $ctxt 182 $cc 183 $acr 200 00$aMolecular biology techniques$b[electronic resource] $ean intensive laboratory course /$f[edited by] Walt Ream and Katharine G. Field 210 $aSan Diego, CA $cAcademic Press$d1998 215 $a1 online resource (245 p.) 300 $aIncludes index. 311 $a0-12-583990-1 327 $aFront Cover; Molecular Biology Techniques: An Intensive Laboratory Course; Copyright Page; Contents; Preface; Course Synopsis; Introduction; Safety Precautions; Daily Schedule; Acknowledgments; Exercises I. DNA Preparation, Polymerase Chain Reaction, and Molecular Cloning; A. Cesium chloride-ethidium bromide density gradient centrifugation; B. PCR to synthesize virD2 flanked with restriction sites; C. Restriction digests of plasmid pGEX2 and PCR products; D. Purification of DNA fragments from agarose; E. Ligation of PCR product to pGEX2 vector 327 $aF. Transformation of E. coli with the ligated plasmidG. Small-scale preparation of plasmid DNA by the alkaline lysis method; H. Restriction analysis; Study questions; Exercises II. Protein Expression, Purification, and Analysis; A. Expression and purification of a fusion protein; B. SDS-polyacrylamide gel electrophoresis; C. Silver stain detection of proteins; D. Western blot (immunoblot) detection of proteins; Study questions; Exercises III. Oligonucleotide-Directed Mutagenesis; A. Restriction digests of virD2 (in pCS64) and pUC119; B. Purification of DNA fragments from agarose 327 $aC. Ligation of restriction fragment and vectorD. Transformation of E. coli with the ligated plasmid and recovery of clones; E. Small-scale preparation of plasmid DNA from broth cultures; F. Restriction digest of DNAs: Examination to confirm insert; G. Preparation of single-stranded DNA template; H. Phosphorylation of oligonucleotide; I. Annealing mutant oligonucleotide to template; J. In vitro DNA synthesis by primer extension; K. Transform synthesis reaction into E. coli DH5a; L. Small-scale preparation of plasmid DNA; M. Confirmation of mutants by restriction analysis; Study questions 327 $aExercises IV. DNA SequencingA. Polyacrylamide sequencing gel electrophoresis; B. Dideoxy sequencing; C. Automated sequencing; D. Introduction to databases and gene sequence analysis; Study questions; Exercises V. Southern Blot Detection of DNA; A. Preparation of genomic DNA from Agrobacterium tumefaciens; B. Restriction digestion of genomic DNA; C. Agarose gel electrophoresis of restriction fragments; D. Southern blot: Denaturation and blotting of DNA; E. Preparation of probe by nick translation; F. Hybridization and washing of Southern blots; Study questions 327 $aExercises VI. Northern Blot Detection of mRNAA. Preparation of RNA from tobacco leaves; B. Agarose-formaldehyde gel electrophoresis; C. Northern blot: Denaturation and blotting of RNA; D. Probe preparation; E. Hybridization and washing of Northern blots; Study questions; Exercises VII. Protein Interaction Analysis in Yeast; A. Yeast transformation; B. Filter ß-galactosidase assay; Index 330 $aThis manual is designed as an intensive introduction to the various tools of molecular biology. It introduces all the basic methods of molecular biology including cloning, PCR, Southern (DNA) blotting, Northern (RNA) blotting, Western blotting, DNA sequencing, oligo-directed mutagenesis, and protein expression.Key Features* Provides well-tested experimental protocols for each technique* Lists the reagents and preparation of each experiment separately* Contains a complete schedule of experiments and the preparation required* Includes study questions at the end of each ch 606 $aMolecular biology$vLaboratory manuals 606 $aMolecular genetics 615 0$aMolecular biology 615 0$aMolecular genetics. 676 $a572.8/078 701 $aField$b Katharine G$01545395 701 $aReam$b Walt$01545396 801 0$bMiAaPQ 801 1$bMiAaPQ 801 2$bMiAaPQ 906 $aBOOK 912 $a9910784526803321 996 $aMolecular biology techniques$93800306 997 $aUNINA