Extracción de ADN genómico / Método NaI
Es un requisito principal obtener ADN genómico macromolecular a partir de sangre completa, cultivos celulares y cultivos tisulares para los análisis genéticos de los genomas y de otros procedimientos en biología molecular. Una variedad de los procedimientos de purificación del ADN implican procesos peligrosos como la extracción con fenol/cloroformo, o son procedimientos muy lentos e ineficientes, como la diálisis prolongada y la transferencia de ADN de un tubo a otro
El extractor WB de ADN de Wako emplea un procedimiento simple de extracción que no es peligroso para la purificación del ADN después de la lisis celular. Este procedimiento, que usa yoduro de sodio (NaI) como agente caotrópico, logra el aislamiento del ADN intacto de alta pureza y de alta producción. La extracción se realiza mediante varias centrifugaciones breves en un solo tubo de microfuga.
Reagents (50 250 Assays)
Note : During the storage, In such a Case, Enzyme Reaction Solution an the NaI Solution may crystalizeout, solubilize the precipitate completely by warming the solution at 50 .
The procedure includes three major steps : 1) Isolation of nucleus fraction. 2)Liberation of DNA, 3) Purification of DNA.
In the first step, cells are lysed by a non-ionic surfactant, polyoxyethylene (10) oxyphenyl ether, followed by a brief centrifugation. By repeating this operation, the majority of cellular macromolecules, including RNAs, are removed from the nucleus fraction. In the next step, the nucleus fraction is treated with protease in the presence of 1 % sodium dodecyl sulfate for 1 hour. By this treatment DNA packed in the nucleus is expanded and liberated from nucleus protection. Simultaneously, protein in the solution is digested into polypeptide. In the final step, the resultant solution is subjected to NaI extraction of DNA as described in literature. Polypeptide and other biological molecules remain soluble in a high concentration of Nal, a chaotropic salt, even after isopropanol is added to the solution to precipitate the nucleic acid.
Store at 2-10°C
Protease is reconstituted by adding 0.6 ml of distilled deionized water. Protease solution reconstituted can be stored at 20.
1- 0.5 ml of whole blood, prepared with EDTA-Na2 as anti-coagulant, is dispensed into a 1.5 ml microcentrifugi tube with screwed cap and then placed standing on ice.
2- After adding 0.5 ml of Lysis Solution and capping the tube, invert the tube several times to genth mix the solution.
3- After a brief centrifugation (10,000 xg. 20 seconds at 4 ), carefully remove the supernatant, keeping the dark red precipitate intact.
4- Add 1 ml of Lysis Solution to the tube and set the capped tube on a microfuge tube mixer for 30seconds at a moderate speed.
5- Remove the supernatant from the tube after a brief centritugation of the mixture at 10,000 g for 20 second at 4.
6- Repeat operation 4 5.
7- Suspend the resultant pellet in 200 l of Enzyme Reaction Solution. Add 10 l of Protease Solution to the suspension and mix gently by inversion.
8- Incubated at 37 for 1 hour. (During the incubation, mix the solution several times by inversion).
9- After the 1 hour-incubation, add 0.3 ml of Nal Solution to the mixture and mix well by inversion.
10- Add 0.5 ml of isopropyl alcohol to the mixture and mix well until DNA, a whitish materisl, appears.
11- After centrifugation at 10,000 xg for 10 minutes at room temperature, gently removed the resultant supernatant. Put the tube upside down to remove the solution remaining on the surface of the tube.
12- Rinse the pellet in the tube by adding 1 ml of Washing Solution (A) to the tube followed by centrifugation (10,000 xg, 5 minutes). Mix thoroughly so that the pellet is removed from the tube wall.
13- Repeat the same operation described above using Washing Solution (B) instead of Washing Solution (A).
14- The resutant pellet is vacuum-dried for about 3 minutes. (Drying should be made within 3 minutes because DNA completely dried may be hard to be dissolved).
Culture cell pellet (103 106cells) such as HeLa and SQ-5, collected in a 1.5 ml microfuge tube is suspended in 1 ml Lysis Solution and then set the capped tube in a microfuge tube mixer.
Follow with operations through 5-14 as described in DNA Extraction Procedure from Whole Blood
Genomic DNA was extracted from 100 l of whole blood with the DNA Extractor WB Kit using smaller volumes of reagents than the standard procedure. In these procedures, 1 5 or 1 4 of the original reagent volumes were employed, Microfuge tubes were 0.6ml. As a reference, genomic DNA was extracted from 500 l of whole blood using the standard procedure