The proteins can then be removed from the antibodies and separated using gel electrophoresis techniques. A very common technique often used is called two-dimensional gel electrophoresis. A sample is run on a very thin strip of polyacrylamide then placed under a perpendicular current, moving the proteins within the sample first in one direction, then separating them in another. This allows separation of molecules by size and by differing charge of molecules of similar molecular weights. Most useful to the fields of biochemistry and molecular genetics is the use of these methods in gene identification.
This is accomplished by subjecting the fragmented DNA to an electrical charge after it has been placed onto an agarose gel plate. Due to differences in length, the DNA restriction fragments will be separated in the gel plate. Another useful tool for scientists has been the DNA probe. A DNA probe is a piece of DNA that binds to certain sequences of the hosts DNA (Devore, 1998). The probe is able to do this because the DNA strand of the probe only binds to the appropriate DNA with a complementary sequence.
There are four important steps in DNA extraction protocol of animal tissue which are enzymatic digestion of cellular protein, DNA precipitation, DNA washing, and DNA hydration (refer to Figure 1 in Appendix 1). Firstly, the cellular protein is digested through enzymatic process. In this step, the animal tissue is grind until it is tiny enough. This is important to break up the cell wall since DNA is located within the nucleus in eukaryotic organisms. Then, 600µl of CTAB buffer is pipetted into the eppendorf tube and mixed well with the grind tissue.
Gel electrophoresis is a procedure used in laboratories to separate DNA, as well as RNA and proteins. A gel slab is placed in a buffer-filled box and an electrical field is applied. The negatively charged DNA will migrate towards the positively charged side, where it can then be recorded and further analyzed. An example for the use of gel electrophoresis would be in identifying people. DNA is present in almost every cell of our body.
Restriction fragment length polymorphism (RFLP) analysis is a technique in which DNA regions are digested using restriction endonucleases, and subjected to radioactive complementary DNA probes to compare the differences in DNA fragment lengths between individuals. The DNA in question is digested using restriction endonuclease(s). The DNA is then run on a gel and appears to be very long. The gel is subject to a chemical that causes the double-stranded DNA to separate into to individual strands. The strands are then transferred to a nylon membrane with using an electric current, where it will bind.
A nuclease is any enzyme that cuts the phosphodiester bonds of the DNA backbone, and an endonuclease is an enzyme that cuts somewhere within a DNA molecule. In contrast, an exonuclease cuts phosphodiester bonds by starting from a free end of the DNA and working inward. Restriction enzymes were originally discovered through their ability to break down, or restrict, foreign DNA. Restriction enzymes can distinguish between the DNA normally present in the cell and foreign DNA, such as infecting bacteriophage DNA. They defend the cell from invasion by cutting foreign DNA into
In the preparation of agarose gel, agarose powder will be mixed with buffer. Agarose powders need to be weighed first before mixing it with buffer. When 1 gram agarose gel is added, it will be followed by 100 ml of buffer. The ratio is 1:100 for agarose powder and buffer respectively. Then, the solution of agarose powder and buffer is put in the microwave in order to melt the agarose until the solution become clear (Carson & Robertson, 2005).
Helicase unwinds the double strand, then organic bases line up next to the corresponding bases on the DNA. It then peels away from the DNA and exits through pores in the nuclear envelope into the cytoplasm. Once in the cytoplasm it attaches to a ribosome, which causes amino acids to assemble in the right order. The DNA then winds back up into its original shape. Transfer RNA helps the amino acids to assemble along the strand.
Rice, G. (n.d.). Dna extraction. Retrieved from http://serc.carleton.edu/microbelife/research_methods/genomics/dnaext.html DNA extraction is removing the DNA from the cells or virus that it is originally from. In order to extract the DNA the cell must break open or lyse. Three instruments used for DNA extraction are a bead beater, centrifuge, and a gel box.
Ligation of EGFP Insert to Plasmid Vector Ligating the EGFP cDNA into a pET41a (+) plasmid in order to create recombinant expression plasmids and run these ligations through gel electrophoresis to visualize the DNA and check the success of the ligations. Five ligation reactions were generated, two actual ligations and three controls, with a total final volume of 20uL each. NcoI and NotI are restriction endonucleases whose purpose are to reduce non-recombinant plasmids from forming and to prevent undesired rearrangements during ligation. Ligation one was a 1:1 molar ratio pET-41a (+) vector: egfp insert that used 50ng NotI/NcoI cut pET-41a (+) DNA, 7ng egfp insert DNA, 1uL of DNA ligase, and the proper quantity of water to dilute 10x ligase buffer to a 1x final concentration. Ligation two was a 1:3 molar ratio pET-41a (+) vector: egfp insert made up of 50ng NcoI/NotI cut pET-41a (+), 21ng egfp insert DNA, 1uL of DNA ligase, and the proper quantity of water to dilute 10x ligase buffer to a 1x final concentration.