DNA Extraction from Fruits PDF

DNA Extraction from Fruits DNA Extraction / isolation : is the process of purification of DNA from sample using a combination of physical and chemical methods. Principle: 1- Breaking the cells, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achiev...

DNA Extraction from Fruits DNA Extraction / isolation : is the process of purification of DNA from sample using a combination of physical and chemical methods. Principle: 1- Breaking the cells, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by chemical and physical methods such as blending, grinding or sonicating the sample. 2- Removing membrane lipids by adding a detergent or surfactants which also serves in cell lysis. 3- Removing proteins by adding protein precipitation solution ( NaCl Saturated solution ). 4- Removing RNA by adding an RNase (almost always done). 5- DNA purification from detergents, proteins, salts and reagents used during cell lysis step. Most commonly used procedure is: [ Ethanol precipitation usually by ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together forming a white glop or giving a pellet upon centrifugation ]. 6- After isolation, the DNA is dissolved in DNA hydration solution, usually in the TE buffer, or in ultra-pure water. 7- Determination of DNA concentration and purity using UV -transilluminator. Procedure: 1- Piece of fruit (1 gm) + 5 ml lysis solution ( 10% SDS + 1% NaCl ). 2- Transfer contents to sugar tube and incubate 15 min @ 65 0C. 3- Filter the content to get rid of the residual debris and transfer 300µl of filtrate to eppendorf. 4- Add 1.5µl RNase solution and mix by inverting 25 times, incubate mixture for 15 min @ 37 0C and then incubate for 1 min on ice (enhance protein precipitation). 5- Add 100µl of protein precipitation solution and vortex vigorously for 20 sec. 6- Centrifuge the mixture for 5 min @ 12,000 rpm.  7- In clean eppendorf, pipette 300µl ice cold-isopropanol and carefully pour the supernatant from previous step to the isopropanol (removal of water shield i.e. dehydration of DNA and its precipitation). 8- Mix the mixture by inverting gently (50 times), until DNA is visible as threads or clump. 9- Centrifuge the mixture for 1 min @ 12,000 rpm, the DNA may be visible as a small white pellet. 10- Carefully discard the supernatant and drain the tube by inverting on clean piece of absorbent paper taking care the pellet remains in the tube. 11- Add 300µl of ice cold 70% ethanol and invert the tube several times (wash DNA pellet) 12- Centrifuge the mixture for 1 min @ 12,000 rpm. 13- Carefully discard the supernatant and drain the tube by inverting on clean piece of absorbent paper taking care the pellet remains in the tube, allow pellet to air dry for 5 sec. 14- Add 100µl DNA hydration solution and vortex 5 sec. 15- Incubate the mixture @ 65 0C for 5 min (dissolve DNA). 16- Incubate DNA @ RT. 17- Dilute 100 or 200 times depend on pellet size. 18- Determine DNA concentration and purity by measuring absorbance at 260 and 280 nm. Calculations: DNA conc. (mg/ml) = [A260 x DF x 50µg/ml]/ 1000 DNA purity= A260/ A280 *Pure DNA 1.7-1.9 (< protein contamination, > RNA contamination) 19- Store DNA @ 2-8 0C in DNA hydration solution.