By M. P. O’Connell, U. Priefer, N. Datta, M. E. Nugent (auth.), Alfred Pühler, Kenneth N. Timmis (eds.)
The improvement of strong new thoughts and refmements of tech niques in molecular genetics in recent times, and the surge in curiosity in biotechnology according to genetic equipment, have heralded a brand new golden age in molecular genetics, and inspired in various disciplines a lot curiosity within the applied sciences themselves and their capability makes use of in easy and utilized biomedical sciences. even if a few very good professional laboratory manuals (especially the chilly Spring Harbor Laboratory manuals through I. H. Miller; R. W. Davies et al. ; and T. Maniatis et al. ) on convinced chapters of molecular genetics exist, no normal textual content that covers a extensive spectrum of the sub ject has to this point been released. the aim of this guide is to pre despatched so much, even though of necessity now not the entire vital equipment of molecular genetics, in a sequence of straightforward experiments, lots of that are simply comprehensive via the microbiologist, biochemist or biotechnologist that has had in simple terms constrained publicity to genetics. the rest of the experiments require both higher familiarity with the topic, or tips via anyone with such adventure. The ebook may still, for this reason, not just allow participants to obtain new proce dures for ongoing tasks, but in addition function a foundation for the educating of molecular genetic innovations in formal predoctoral and postdoctoral laboratory courses.
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Extra info for Advanced Molecular Genetics
8 Simplified Alkaline Extraction Procedure (Ish-Horowicz and Burke 1981) Centrifuge 1 ml of an overnight bacterial culture. 0, 10 mM EDTA; incubate 5 min at room temperature. 2 N NaOH, 1% SDS, mix gently, put on ice for 5 min. 8, mix gently, leave on ice for 5 min. Remove precipitate by centrifugation for 1 min. Add,2 vol. EtOH to the supernatant, hold 2 min at room temperature, precipitate DNA by centrifugation. Wash pellet with 70% EtOH. Drain and dry pellet, resuspend in 50 pI buffer. Note: To achieve higher sensitivity to restriction enzymes, one can insert a phenol extraction step before precipitating DNA with EtOH.
5. Experiment 4: Comparisons of Plasmids in Wild Strains and in the E. coli K12 Recipients 6. Experiment 5: Classification of Unknown Plasmids According to Incompatibility (Inc) Group. . . . . . . . . . . . . . . . . . . . . . . a) Introduction. . . . . . . . . . . . . . . . . . . . .. b) Objective. . . . . . . . . . . . . . . . . . . . . .. c) Procedure . . . . . . . .
A-DNA restriction fragments. A-DNA restriction fragments produced by cleavage with EeoRI, HindIII, and by EcoRI/HindIII double digestion. The sizes are given in bp. The full-size phage genome is determined to be 49 kb (Phillipsen et al. 320 930 880 12: 590 13: 100 Characterization of Plasmid DNA by Agarose Gel Electrophoresis 35 b) Objectives DNA from plasmid pBR325 is cleaved with several restriction enzymes. Digestion with EeoRl, Hin dIll , Bam HI , and PstI, which each have single cleavage sites on pBR325, yield linear DNA molecules; double digestions with these enzymes result in the production of two fragments each.