Only to purify Plasmid
Mini-Prep procedure is used to isolate small plasmid DNA from bacteria while limiting contaminating proteins and genomic DNA. The plasmid quality is acceptable for restriction analysis, sequencing, cloning, or other purposes.
The Monarch Plasmid Miniprep Kit is a rapid and reliable method for the purification of up to 20 μg of high quality plasmid DNA. (Protocol -tips).
The kit consist on the following steps:
- PELLET - Spinning them fast to the cells sink and liquid media stays on top
- RESUSPEND - resuspend cells in first buffer
- LYSE Cell lysis or cellular disruption is a method in which the outer boundary or cell membrane is broken down or destroyed in order to release inter-cellular materials such as DNA, RNA, protein or organelles from a cell.
- NEUTRALIZE -
- BIND - Binds DNA to spin column (mini tube)
- WASH - to remove any last proteins, to keep the wanted DNA
- ELUTE - Add a little salt to clean the DNA off the column and put it into a new tube
**QUBIT : Not from the Kit but a very important step. This will help determine the concentration, yield and purity of a DNA sample. - Needed for QUBIT:
- MASTER MIX ------> (Buffer + Regeant ) **Need to do calculations based on the amount of samples
- Qubit Assay tubes - they do not absorb UV light . Must be used to get an accurate reading from the Qubit machine
The term ‘plasmid’ was coined by Joshua Lederberg in 1952. Originally evolved from bacteria, plasmids are extrachromosomal genetic elements present in most species of Archae, Eukarya and Eubacteria that can replicate independently. Plasmids are circular double stranded DNA molecule that are distinct from the cells chromosomal DNA.
The structure and function of a bacterial cell is directed by the genetic material contained within the chromosomal DNA. In some cases plasmids are generally not essential for the survival of the host bacterium. Although not essential, plasmids contribute significantly to bacterial genetic diversity and plasticity by encoding functions that might not be specified by the bacterial chromosomal DNA. Plasmids specify traits that allow the host to persist in environments that would otherwise be either lethal or restrictive for growth. For example antibiotic resistance and protein expression. Antibiotic resistance genes are often encoded by the plasmid, which allows the bacteria to persist in an antibiotic containing environment, thereby providing the bacterium with a competitive advantage over antibiotic-sensitive species. As a tool, plasmids can be modified to express the protein of interest (e.g., production of human insulin using recombinant DNA technology).
Plasmids have served as invaluable model systems for the study of processes such as DNA replication, segregation, conjugation, and evolution. Plasmids have been pivotal to modern recombinant DNA technology as a tool in gene-cloning and as a vehicle for gene-expression.
Characteristics of Plasmid - Plasmids present in the bacterium differ in their physical properties such as in size (kbp), geometry and copy number.
Plasmid Size - Plasmids range in size from 1 kbp (kilo base pair) to 1000 (kilo base pair) megaplasmids that are many hundred base pairs in size.
Plasmid Geometry -Although most plasmids possess a circular geometry, there are now many examples of plasmids that are linear in a variety of bacteria. Plasmid DNA may appear in one of the five conformations nicked open circular DNA which has one strand cut, relaxed circular DNA is fully intact with both strands uncut, but has been enzymatically relaxed, linear DNA has free ends, supercoiled DNA is fully intact with both strands uncut, and supercoiled denatured DNA is like super coiled DNA, but has unpaired regions that make it slightly less compact.
Plasmid Copy Numbers - Copy number refers to the average or expected number of copies per host cell. Plasmids are either low, medium or high copy number. Knowing which category plasmid falls under is very important when starting out an experiment. If working with a low-copy number plasmid which is associated with a low yield and might therefore be required to set up more cultures. On the other hand, if a poor yield is obtained from a high copy plasmid, troubleshooting is required. In bacterium with high copy number plasmids, during cell division the plasmids get segregate randomly in the daughter cells, whereas bacterium with low copy numbers, during cell division and partition the plasmids divided equally in the daughter cells. An advantage of high copy number is the greater stability of the plasmid when random partitioning (i.e. partitioning of plasmids into daughter cells) occurs at cell division.
Plasmid Isolation - The isolation of plasmid DNA from bacteria is a crucial technique in molecular biology and is an essential step in many procedures such as cloning, DNA sequencing, transfection, and gene therapy. These manipulations require the isolation of high purity plasmid DNA. The purified plasmid DNA can be used for immediate use in all molecular biology procedures such as digestion with restriction enzymes, cloning, PCR, transfection, in vitro translation, blotting and sequencing.
Alkaline lysis is a method used in molecular biology, to isolate plasmid DNA or other cell components such as proteins by breaking the cells open. Bacteria containing the plasmid of interest is first grown, and then allowed to lyse with an alkaline lysis buffer consisting of a detergent sodium dodecyl sulfate (SDS) and a strong base sodium hydroxide. The detergent cleaves the phospholipid bilayer of membrane and the alkali denatures the proteins which are involved in maintaining the structure of the cell membrane. Through a series of steps involving agitation, precipitation, centrifugation, and the removal of supernatant, cellular debris is removed and the plasmid is isolated and purified.
Sources:
https://www.mybiosource.com/learn/testing-procedures/plasmid-isolation
