Midi, Maxis, Megas, and Monsters Oh My!!!

Comparison Chart N.A. Horn et al., Cancer Gene Therapy Using Plasmid DNA- Purification of DNA for Human Clinical Trials, Human Gene Therapy, 6: 565-573, 1995. R.M. Conry, A Carcinoembryonic Antigen Polynucleotide Vaccine for Human Clinical Use, Cancer Gene Therapy, 2: 33-38, 1995. Z. J. Ren et al., Cloning Of Linear DNAs In Vivo By Over- expressed T4 DNA Ligase Construction Of A T4 Phage Hoc Gene Display Vector. GENE, 195:303-311, 1997. M.V. Chudaev, S.A. Usanov, Expression Of Functionally Active Cytochrome b(5) In Escherichia coli: Isolation, Purification And Use Of The Immobilized Recombinant Heme Protein For Affinity Chromatography Of Electron-Transfer Proteins, Biochemistry-Moscow, 62:401-411, 1997. B. He et al., Rapid Mutagenesis and Purification of Phage RNA Polymerases, Protein Express Purif, 9: 142-151, 1997. K.S. Kim et al., A Ti Plasmid Encoded Enzyme Required For Degradation Of Mannopine Is Functionally Homologous To The T- Region-Encoded Enzyme Required For Synthesis Of This Opine In Crown Gall Tumors, J. Bacteriol, 178: 3285-3292, 1996. S. Rivas et al., TrwD A Protein Encoded By The Inc W Plasmid R388 Displays An ATP Hydrolase Activity Essential For Bacterial Conjugation, J. Biol. Chem, 272:25583-25590, 1997. J.S.M. Tschen et al., Rhizoctonia Solani Incited Damping-Off In Radish Suppressed By Plasmid And Non-Plasmid Carrying Strains Of R-Solani That Enhance Host Chitinase Activity, J. Phytopathology, 145:363-369, 1997. M.M. Sikorski, Expression Of Lupinus Luteus cDNA Coding For PR10 Protein In Escherichia Coli: Purification Of The Recombinant Protein For Structural And Functional Studies, Acta Biochim Pol, 44: 565-578, 1997 V. Escriou et al., Triple Helix Formation On Plasmid DNA Determined By A Size-Exclusion Chromatographic Method, Anal Biochem, 248:102-110, 1997. V. Tedeschi et al., A Specific Antibody Response To HCV E2 Elicited In Mice By Intramuscular Inoculation Of Plasmid DNA Containing Coding Sequences For E2, Hepatology, 25:459-462, 1997 W.L. Picking et al, Cloning, Expression And Affinity Purification Of Recombinant Shigella flexneri Invasion Plasmid Antigens IpaB And IpaC, Protein Express Purif, 8:401- 408, 1996 I. Ohsugi et al., Expression Of Mouse Uterine Peptidyl Arginine Deaminase In Escherichia-coli Construction Of Expression Plasmid And Properties Of The Recombinant Enzyme, Arch. Biochem. Biophys. 317: 62-68, 1995. G.V. Gololobov et al., Cleavage of Supercoiled Plasmid DNA By Autoantibody FAB Fragment Application of the Flow Linear Dichroism Technique, Proc. Nat. Acad. Sci., 92:254-257, 1995 P. Arca et al., Plasmid-Encoded Fosfomycin Resistance In Bacteria Isolated From The Urinary Tract In A Multicentre Survey, J. Antimicrob Chemoth, 40: 393-399, 1997 W.F. Vann et al., Purification And Characterization Of The Escherichia Coli K1 Neub Gene Product N-Acetylneuraminic Acid Synthetase, Glycobiology 7:697-701, 1997. M. Nagao et al., Secretory Production Of Erythropoietin And The Extracellular Domain Of The Erythropoietin Receptor By Bacillus brevis: Affinity Purification And Characterization, Biosci Biotech Bioch, 61: 670-674, 1997. B. MozeticFrancky et al., High-Yield Expression And Purification Of Recombinant Human Macrophage Migration Inhibitory Factor, Protein Express Purif, 9: 115-124, 1997. D.O. Nwankwo, Cloning And Expression Of Aat II Restriction- Modification System In Escherichia coli, GENE, 185: 105-109, 1997. M. Llosa, Functional Domains In Protein Trwc Of Plasmid R388: Dissected DNA Strand Transferase And DNA Helicase Activities Reconstitute Protein Function, J Mol Biol, 264: 56-67, 1996. R. Hanai et al., Molecular Dissection Of A Protein SopB Essential For Escherichia coli R Plasmid Partition, J. Biol Chem, 271:17469-17475, 1996. M. Larsson, A General Bacterial Expression System for Functional Analysis of cDNA-Encoded Proteins, Protein Express Purif, 7: 447-457, 1996. E.P. Johnson et al., Plasmid RK2 Toxin Protein ParE: Purification And Interaction With The ParD Antitoxin Protein, J. Bacterial, 178: 1420-1429, 1996. M. Weber, Effects Of Lipopolysaccharide On Transfection Efficiency In Eukaryotic Cells, Biotechniques, 19: 930, 1995 I have a vague recollection of my advisor once telling me "I used to make and purify my own restriction enzymes!" I think we all have an equivalent "walking to school in the snow" story that we draw upon to make the point that lab life has become much better since the days "we did our time". But the fact is, there was a time when everything in the lab was done in scale and we did everything ourselves, including preparing every buffer that was necessary. This was a time when we never quite knew how much we were going to need or how many times experiments would need to be repeated. The majority of the early procedures simply needed more material since assays or techniques did not have the sensitivity of those currently available. This was science's more-is-better era. Times seem to have changed. Everything we do in the lab, and indeed many of the products that are commercially available, seem to be related to speed, sensitivity, and allowing us to skate by with just the right amount of material. Much of this focus is appropriate since many of the procedures now performed in the laboratory are screening procedures. Even the wave of new instruments provides us with the ability to work with increasing speed, minimal experimental materials, and greater sensitivity such that working in large scale simply isn't needed. Still, however, a number of procedures are routinely, and out of necessity, done in scale. One of the procedures that immediately comes to mind for most molecular biologists is plasmid preparation. While miniprep procedures are geared for screening or rapidly gathering sequence information, midi- and maxi-preps are required to provide significant microgram quantities of plasmid that are essential for repeating and data-gathering experiments. In this regard, speed and throughput are generally not major issues; rather the quality of the preparation becomes the paramount factor. Historically, and for the most part still today, the gold standard for large-scale plasmid preparation quality has been to run a plasmid through one or two cesium chloride centrifugations. Although the time needed for centrifugation was lengthy, numerous experiments documented that the purity of a sample obtained was significantly superior to more expedient preparation procedures. Transfection efficiencies, for example, could be several-fold higher with plasmids prepared by density gradients; when working with rather rare events, any incremental gain was experimentally significant. As with other laboratory techniques, there have been some remarkable improvements in larger-scale plasmid purification methods that have been brought about by exploiting unique matrices or reagents. Many commercial products, in fact, now approach the quality of cesium chloride gradients and are in a format that can be completed in less then half a day, rather than days with centrifugation-based protocols. These products provide a laboratory with quality, while minimizing time expenditures. Additionally, most procedures are "waste-friendly"; that is, they do not generate hazardous wastes, such as phenol-chloroform or cesium chloride/ethidium-bromide. Sounds great, you say. Well, it gets even better! Many of the maxi protocols can handle enormous quantities of starting cultures and high plasmid concentrations (i.e., they do not become overloaded). When you consider time, material, quality, and waste, in the long run these kits are significantly less expensive than you might think. When deciding which product is best for your lab, you must keep in mind what your specific application is and if there are typical culture volumes that your laboratory processes. If the primary interest is in labeling probes, for example, your preparation may not require the absolute "cesium standard" of some products. Similarly, consider the size of the routine plasmid preparations performed in the laboratory. Some kits have a standardized volume of starting culture; if your laboratory routinely processes culture sizes smaller than those, you may be throwing money down the drain. Conversely, for larger cultures, you may not be achieving the necessary yield. Many of the current products are considered "scaleable" in that only the amount of material that is needed to match your culture is necessary. This feature provides a certain amount of flexibility and cost effectiveness. Many different formats of midi- and maxi-preps are available. Which format is better is a factor for you to consider based on available laboratory equipment, yield, application, and personal preferences. It is best to examine the stated parameters of performance for each product and weigh these against your preference of format and specific application. You may find that for the range of applications of plasmids in your laboratory, it might be advisable to stock a variety of kits. These kits provide useful, expedient alternatives for your lab. As significant as miniprep kits have become to the laboratory, these large-scale cousins have earned a spot on the shelf as well. Of course, one of the choices that you still have to make is whether to provide your grad student or post-doc the opportunity of having his or her own "walk to school in the snow" stories that they can pass on to future generations of scientists. Comparison Chart The author can be reached at: tfunger@aol.com.

Midi, Maxis, Megas, and Monsters Oh My!!!

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