Isolation of DNA
Isolation of DNA in plants is done using daunNeomarica longifolia young. Neomarica longifoliayang young leaves are used because the leaves are chloroplasts containing DNA and the leaves are still young, yet many cell wall and easily purified (The University of Utah 2005: 1).
Isolation of DNA can also be done in yeast. Yeast are unicellular fungi that occupy the habitat of liquid and moist. Yeasts reproduce asexually by simple cell division or by releasing stem cells from stem cells. Some yeasts also reproduce sexually, by way mementuk actionable or basidia (Campbell et al. 2003: 193).
Yeast used in lab adalahCandida parapsilosis DNA isolation 24 hours old. Candida parapsilosis is a normal inhabitant of the human epithelial tissue moist. Certain circumstances may cause Candida parapsilosis be pathogenic to overgrow and liberate hazardous substances. Age leavened 24 hours due to these microorganisms have had more DNA (Campbell et al. 2003: 194-195).
Isolating DNA is done in several stages. The first phase is the network isolation. The isolation of the blood system is done by inserting a 3 mL of blood into a centrifuge tube (Boyer 1993: 454). Isolating DNA in plants is done by mengerus plants until smooth to get a plant cell to be isolated DNA (The University of Utah 2005: 1). The isolation of yeast is done by separating the yeast with the medium (Duncan et al. 2004: 1).
The next stage is the extraction of DNA. DNA extraction was done by lysing cells and purifying DNA impurities from substances derived from the cells. Substances that become impurities in general are protein, plosakarida in plants, the compounds (Invitrogen 2003: 2).
Blood DNA extraction is done by adding a solution pelisis red blood cells and diinvert. The solution serves to destroy the red blood cells. Invert is shaking on the tube with a figure eight pattern with the aim to accelerate the solution is evenly distributed and collisions between fast blood cells so that the lysis (Boyer 1993: 454). The composition of the solution pelisis red blood cells are EDTA, NH4Cl, KHCO3, and KOH. EDTA serves to prevent damage to the DNA as in the cell and the environment around the cell there are many enzymes that can damage DNA. The addition of EDTA solution was also performed on DNA extracted yeast (Boyer 1993: 455). DNA extraction plant is done by the addition of as much as 5 mL extraction buffer that has been heated. The addition of buffer solution aims to lyse cell walls in plants (Invitrogen 2003: 3). Subsequent tube was incubated for 10 minutes. Incubation aims to keep the temperature optimal (Cell Biology Research 2004: 1).
The tubes are incubated further centrifuged. The working principle of centrifugation related to the mass, density, and parts of structures or molecules are centrifuged and related to the mass, density and viscosity of the solution. Based on molecular weight, it will produce two sections that are easy to separate. It consists of two parts supernatant and pellet. Supernatant is part of the molecular weight is light and at the top of the tube while the pellets are part of the heavier molecular weight and are at the bottom of the tube. Before centrifugation, the tube is closed beforehand using parafilm. This is done to prevent spilling the contents of the tube when centrifuged (Boyer 1993 191-196).
The resulting supernatant on blood centrifugation process is the red blood cells that have been lysis. Pellets produced from centrifugation of blood are white blood cells that settles. The resulting supernatant the centrifuge process of plant DNA is the cell wall lysis. The resulting pellets are cell nucleus contains DNA. The resulting supernatant the centrifugation process yeast is a DNA contained in the liquid pellets produced are part of the cell buildup. Centrifugation at yeast DNA isolation only one that has been generating DNA. Interest centrifugation process is to separate the DNA from other parts of the cells (Mader 1993: 64).
Tubes that have been centrifuged in blood DNA isolation, the supernatant discarded and then peletnya divorteks. Vortex is the process of flipping back the tube. The function of the vortex is menghomogensikan solution so that white blood cells do not clump at the bottom of the tube (Boyer 1993: 456). The next stage, the pellets pelisis added a solution of white blood cells and the mixture is removed by using a pipette. The purpose of the addition of a solution pelisis white blood cells are white blood cells lyse membranes so that the pores of the cell will swell causing white blood cells rupture and DNA will be out. The composition of the white blood cells pelisis solution is Tris-HCl, SDS, EDTA. Tris-HCl serves to reduce the disulfite bonds of the protein so that the protein to be separated from white blood cells. SDS serves to dissolve the protein on the membrane of white blood cells and EDTA serves to protect DNA from DNAse activity so that the DNA is not damaged (Sinex 2004: 2). Pipetting in issuing need to be done quickly to prevent clotting (Purves & Rybicki 2003: 2).
Centrifugation at isolation of plant DNA is done after the sample is added chloroform isoamyl and divorteks. The function of these additions is to remove protein still contained in the sample. Centrifugation at yeast DNA Isolation conducted after the sample divorteks and diboiling for 20 minutes. Stages boiling has several advantages over other isolation means, namely a fast, cheap and easy to do stage (Duncan et al. 2004: 2).
The next stage after centrifugation at DNA isolation is the purification or purification. Stages in the blood DNA isolation aimed at cleaning the white blood cells from other substances. Tubes were given a solution pelisis white blood cells and removed thereof didiberikan RNase and incubated for 15 min at 37 ° C. It aims to optimize the work of an enzyme that is strongly influenced by temperature. Stages of purification at the plant DNA isolation performed by administering cold absolute ethanol and RNase. Function Award cold absolute ethanol is that separate DNA from other substances and does not destroy DNA (Harley 2005: 409-410). RNase serves to degrade RNA, so the only DNA that looks (Boyer 1993: 455).
The next stage in the blood DNA isolation ie precipitation stage performed by dripping 50 protein precipitation solution and then divorteks. Protein precipitation solution consisting of ammonium acetate if it binds to proteins resulting in the formation of new compounds that have lower solubility, causing the protein to precipitate. The solution is then centrifuged another 15 minutes at a speed of 3000 rpm. Supernatant containing DNA was then poured into a tube containing cold isopropanol and tubes diinvert. Isopropanol Award aims to visualize DNA. Furthermore, the tube was centrifuged for 5 minutes at a speed of 3000 rpm. The results of the centrifugation pellet is the presence of DNA in the bottom of the tube which is then added 70% ethanol and inverted back. Ethanol Award aims to clean-pengotornya DNA from impurities. Once mixed, the tube was then centrifuged for 5 minutes at a speed of 3000 rpm. The end result is that DNA is located on the bottom edge of the tube. The last step is the provision of Tris-EDTA which aims to resolve the DNA to be preserved (Harley 2005: 409-410).
Phase respiritasi on plant DNA isolation was done by pellet was washed with 70% ethanol and the pellet was dried for 30 minutes. Pellets then diluted with Tris-EDTA as much as 100 mL and added RNase. The DNA sample that produced a pellet which is located at the base of the tube. The resulting DNA is stored in TE to keep the structure of DNA and maintain pH. DNA in the yeast DNA isolation generated after centrifugation. The resulting DNA contained in the supernatant which is located at the top of the tube. The DNA also stored in TE
(Invitrogen 2003: 5).
DNA results obtained in the lab shows the same structure as the literature although not clearly visible on the double-stranded structure of DNA. The DNA in the form of a little white blob and very smooth. This is in accordance with that stated in the literature that DNA has a thin and delicate chain. All three samples were used show the same thing (Campbell et al. 2002: 284).
No comments:
Post a Comment