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Article Directory :: Reference & Education Articles
Miniprep Protocol Made Easier - Any study involving the transformation, transfection and DNA cloning cannot happen without the first step of purifying and isolating plasmids. Plasmids are extra-chromosomal DNA found in certain strains of bacteria e.g. E.coli. They have unique features that allow new segments of DNA to be inserted in them; hence can be expressed in bacteria.
The Traditional Miniprep Protocol - The miniprep protocol is a renown method used by most scientists to isolate and purify plasmid DNA from bacterial cultures and hence obtain the pure plasmid for cloning or transfection purposes.
Once a bacterial culture is obtained, it must be lysed and the desired bacterial plasmids isolated from the genomic DNA. Minipreps take the advantage that genomic DNA is quite large as compared to Plasmid DNA and hence, the former can be precipitated using buffers of high salt concentration.
Upon successful precipitation of genomic DNA, the remnant plasmid DNA and RNA can be separated using ribonucleases that degrades the RNA, leaving a pure sample of plasmid DNA.
Cell Lysis as a Prerequisite for Plasmid Purification - The one challenge that scientists face is the difficulty of lysing cells safely without disrupting and shearing the DNA segments. Mechanical methods are normally used but many are not efficient enough to leave the plasmid DNA intact.
Former methods required the use of a long miniprep protocol that would require overnight preparation before use the following day. Scientist had the tedious task of preparing miniprep alkaline solutions that were to be used in various steps of the cell lyses and washing off the supernatants.
Until the ultimate goal of obtaining the plasmid pellets from the bacterial culture is met, it can take approximately 30min to 1 hour depending on speed and expertise. Off course, there are high chances of contaminating the lysate due to handling of the plasmid DNA.
The Solution - Advanced molecular techniques have been developed to ensure the safe lyses of any bacterial cells to produce intact plasmid DNA for cloning. Such methods do not involve many steps that would lead to contamination of the plasmid sample
The recent molecular techniques incorporate all of the many steps of obtaining plasmid DNA into one single step i.e. cell lyses, centrifuging, re-suspending, and washing. In addition, some do not even require washing of the plasmid pellets; a major source of organic contamination to the plasmids DNA
Above all, the hurdle of lysing some resistant types of cells like gram +ve and mycobacterium has been achieved by improved biotechnology approaches to cell lysing techniques, not to mention that the kits are environmentally friendly to use and dispose
Jonathan has extensive experience in the area of DNA Extraction and Isolation, as well as the use of several different Miniprep Protocols. Learn more about the more technologically advanced and convenient Miniprep Protocols discussed in this article by viewing this Miniprep Protocol list.
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