Microscale isolation

C. Morgan and N. Moir,/. Chem. Educ. 73, 1040-1041 (1996). Rapid Microscale Isolation and Purification of Yeast Alcohol Dehydrogenase Using Cibacron Blue Affinity Chromatography. ... 

Microscale Isolation of Plasmid DNA by Alkaline Lysis (Bimboim, 1983)... 

As an alternative to the boiling method, students may perform the microscale isolation method. This version is more convenient to set up and complete, especially if facilities are limited. The procedure works well with unamplified or chloroamphenicol-amplified cultures. Each milliliter of an unamplified, overnight culture will yield approximately 1-2 /xg of plasmid DNA. This isolation procedure requires IV2 to 2 hours of student lab time if a cell culture is prepared in advance. 

Extrachromosomal DNA molecules called plasmids are harbored in some strains of E. coli. The normal copy number of the plasmids is small, between 2 and 10 however, if these strains of E. coli are grown in the presence of chloramphenicol, up to 3000 copies may be replicated per cell. Plasmid DNA has been demonstrated to be a useful vehicle in molecular cloning. This experiment describes a method for the growth of E. coli and amplification of the ColEl plasmids. The plasmids will be isolated from E. coli cells by one of two methods, a large-scale boiling method or a microscale alkaline lysis method. The DNA plasmids will be measured for molecular size by agarose electrophoresis. 

Two isolation procedures based on methods in category 2 are described in this experiment, a large-scale and a microscale method. Each procedure yields plasmid DNA that is sufficiently pure for size analysis by agarose electrophoresis and for digestion by restriction enzymes as described in Experiment 15. 

Most of the ionic chiral auxiliary-mediated reactions presented in Table 4 occur with very respectable ees, even at quite high conversions. The best of these in terms of overall optical and chemical yield is the reaction shown in entry 4, which gives an essentially quantitative GC yield of the 3-lactam photoproduct with an ee of 99%. For a variety of practical reasons, and because modem methods of analysis do not require large sample sizes, all but one of the reactions in Table 4 were carried out on a microscale (< 10 mg). The lone exception was reaction 4, which could easily be scaled up to the 500 mg level to afford an ee of 99% with an isolated chemical yield of 91 % [21 ]. This was done by irradiating the salt... 

A final scale-up isolation may be unnecessary with the microscale approach since modern spectroscopic techniques allow the solving of structures with... 

Transneptunium tris(cyclopentadienyl) complexes can be prepared on a microscale from the reaction of molten Cp2Be with the anhydrous trichlorides (equation 21). The products are isolated by fractional sublimation. X-ray powder diffraction indicates that these CpsAn complexes are... 

All of these investigations allow the easy identification of new tro-panes, when isolated on a microscale, by mass spectrometry. 

However, microscale (matrix isolation spectroscopic) experiments show that the nonsolvated Grignard reagent forms at even lower temperatures (15 K), and no intermediate charge-transfer complex is formed ... 

To detect structural variations in the O-PS structure of A. salmonicida from typical and atypical isolates we have used a microscale CE—MS-based method for analysis of A. salmonicida LPS directly on the bacterial cells. This method involves pretreatment ofbacterial cells ( 10 —10lu cells) with proteinase K, RNase and DNase followed by delipidation with mild acetic acid and subsequent MS analysis. Using this method, we have analyzed LPS structure of 39 typical and atypical isolates of A. salmonicida and related species (Table 1). All A salmonicida strains examined could be divided into three structural patterns, type A, type B, and type C, according to the structure of their corresponding O-PSs (Scheme 2). Majority of typical A. salmonicida isolates belonged to type A displayed the... 

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