بعض الأجهزه المستخدمة في الوراثة الجزيئية Explanation of some equipment and operation ways

Eppendorf tubesdeep well plate.

Micropipettes Pipette

electrophoresis equipment Centrifuge

Techniques in molecular genetics Techniques in molecular genetics mainly can be divided into main classes, separation and purification of DNA, mRNA, and protein, detection using technique of gel electrophoresis, amplification by PCR and agarose and polyacrylamide gels electrophoresis analysis. Genetic materials found very limited quantity in the cell, and then should be amplified to work on it. The best known methods for amplification are Polymerase Chain Reaction (PCR). Expression analysis could be done at mRNA level by Real-Time PCR (RT-PCR), at protein level by SDS-PAGE.

Separation and purification الفصل والتنقية In separation DNA and mRNA are isolated from cells (the separation) using different techniques. Detection اكتشاف Isolated DNA and RNA is run through a gel using technique of gel electrophoresis in order to detect the isolated material. Amplification مضاعفة The main materials used in polymerase chain reaction are DNA nucleotides, template DNA, primers and Taq polymerase.

DNA Extraction DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and two optional steps in a DNA: 1.Breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by chemical and physical methods- blending, grinding or sonicating the sample. 2.Removing membrane lipids by adding a detergent or surfactants. 3.Removing proteins by adding a protease(optional but almost always done) then separated by ultracentrifuge. 4. washing the supernatant ( DNA and RNA) by protease, twice and used ultracentrifuge in each time.

5.Removing RNA by adding an RNase (often done) and used ultracentrifuge. This step repeated twice. 6.Precipitating the DNA with an alcohol usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation.

7.Refinements of the technique include adding a chelating agent to sequester divalent cations such as Mg 2+ and Ca 2+, which prevents enzymes like DNase from degrading the DNA. 8.Cellular and histone proteins bound to the DNA can be removed either by 1- adding a protease or 2- by having precipitated the proteins with sodium or ammonium acetate, or3- extracted them with a phenol-chloroform mixture prior to the DNA-precipitation.

RNA extraction RNA extraction is the purification of RNA from biological samples. This procedure is carried out by adding DNas and seprate by ultracentrifuge. Several methods are used in molecular biology to isolate RNA from samples, the most common of these is Guanidinium thiocyanate-phenol-chloroform extraction.

Another method for extraction of nucleic acids from plant tissues Phenol–chloroform extraction is a liquid–liquid extraction technique. Equal volumes of a phenol:chloroform mixture and an aqueous sample are mixed, forming a biphasic mixture. This method relies on phase separation by centrifugation of a mix of the aqueous sample and a solution containing water- saturated phenol, chloroform and a chaotropic denaturing solution (guanidinium thiocyanate) resulting in an upper aqueous phase and a lower organic phase. Nucleic acid (RNA) partitions in the aqueous phase, while protein partitions in organic phase. In a last step, RNA is recovered from the aqueous phase by precipitation with 2-propanol or ethanol.