Kit de extracción de ADN (Método de yoduro sódico)


Kit de extracción de ADN (Método de yoduro sódico)

Método de yoduro sódico

Para la detección y medición del ADN en líquidos, éste debe aislarse de las proteínas de la muestra. Tradicionalmente, se usa el procedimiento PK-SDS. Este procedimiento emplea la digestión de la proteasa K en presencia de SDS, seguida de extracciones con solventes orgánicos. Aunque con este procedimiento se obtiene ADN de relativamente alta calidad, requiere de un trabajo que lleva mucho tiempo y el uso de solventes tóxicos como el fenol y el cloroformo.

El equipo para la extracción de ADN de Wako utiliza un procedimiento de extracción para la purificación de ADN de suero humano en un solo tubo. Este procedimiento emplea el yoduro sódico (NaI) como agente caotrópico y logra aislar ADN de alta calidad y alta recuperación de los líquidos biológicos sin manipulaciones complejas y laboriosas

Productos Relacionados 


Ref:
Kit de extracción de ADN (Método de yoduro sódico)

DATA

Contents (50 assays)

(1) Sodium Iodide Solution

26 mL x 1

(2) Sodium N-Lauroyl Sarcosinate Solution

1.2 mL x 1

(3) Washing Solution (A)

42 mL x 1

(4) Washing Solution (B)

40 mL x 2

(5) Glycogen Solution

0.1 mL x 1

Materials Needed

Reagents

 

Isopropanol (Special Grade)

45 mL

Distilled Deionized Water

6 mL

 

 

Apparatus

 

Microcentrifuge (Max. 12,000 g)

 

Microfuge tubes with caps (1.5 or 2 mL)

 

Vortex Mixer

 

Principle of DNA Extraction

A high concentration of chaotropic, NaI, and an anionic detergent participate in solubilization of the proteins and lipids contained in biological samples. After addition of isopropanol to the mixture, nucleic acids are co-precipitated with polysaccharide glycogen as a carrier, while other components remain soluble in the solution phase.

Storage:         

Store at 2-10ºC.

DNA Extraction Procedure from Serum

Preparation of Reagents

1.      Before DNA extraction, the following solution is prepared: 26mL of NaI Solution (one bottle) is diluted with 6mL of distilled deionized water. Add 1mL of Sodium N-Lauroyl Sarcosinate Solution and 65µL of Glycogen Solution to the diluted solution and mix well.

2.      Add 2µL of Glycogen Solution to Washing Solution (B) and mix well.

Technical Notes

The distilled deionized water for the dilution of NaI Solution should be free of DNA. The prepared Washing Solution (B) is stable for at least 1 week at 4ºC. When using a small volume of Washing Solution (B), add Glycogen Solution to the appropriate volume in a sterilized tube. During storage, NaI Solution may crystallize. Heat the solution to 50ºC to aid in dissolving.

DNA Extraction Procedure

1.      Dispense 100µL of serum to a sterile, plastic 1.5mL-microcentrifuge tube.

2.      Add 300µL of prepared NaI Solution to the tube and mix.

3.      Incubate the tube in a heating block at 60ºC for 15 minutes.

4.      Remove the tube from the heating block, and add 400µL of isopropanol to the tube and mix well.

5.      After letting the tube stand for 15 minutes at room temperature, centrifuge the tube (10,000 g x 15 minutes).

6.      Remove as much supernatant as possible from the tube. After removal of the supernatant, place the tube upside down on a paper filter to remove residual liquid on the tube wall.

7.      Resuspend the resultant white pellet in 1mL of prepared Washing Solution (B) and swirl until the pellet is detached from the tube wall.

8.      After a brief centrifugation (10,000 g x 5 minutes), discard the supernatant from the tube, and the resulting pellet is vacuum-dried. This pellet contains the DNA for analysis.

Sample Pretreatment for Total DNA Assay in Thresholdä Systems*

Preparation of Reagents

1.      65µL of Glycogen Solution is added to 26mL of NaI Solution (one bottle).

2.      2µL of Glycogen Solution is added to 40mL of Washing Solution (B), followed by immediate mixing.

Technical Notes

The prepared Washing Solution (B) is stable for at least 1 week at 4ºC. When using a small volume of Washing Solution (B), add Glycogen Solution to the appropriate volume in a sterilized tube. During storage, NaI Solution may crystallize. Heat the solution to 50ºC to aid in dissolving. The extraction procedure is performed prior to heat denaturation of the DNA. It is important, therefore, to practice aseptic technique to minimize DNA contamination. Sterile, wrapped tips, sterile tubes, and gloves should be used throughout this procedure.

DNA Extraction Procedure

1.      Dispense 500µL of sample to a sterile, plastic 2mL-microcentrifuge tube with a cap.

2.      Add 20µL of Sodium N-Lauroyl Sarcosinate Solution to the tube and mix.

3.      Add 500µL of NaI Solution containing glycogen to the mixture, vortex and then incubate at 40ºC for 15 minutes.

4.      Add 900µL isopropanol to the mixture, vortex and then let stand at room temperature for 15 minutes.

5.      After a brief centrifugation (10,000 g x 5 minutes), a white pellet may be visible. The resultant supernatant is discarded and the residual solution on the tube wall is removed by placing the tube upside down on filter paper.

6.      Add 800µL of Washing Solution (A) to the tube and vortex vigorously as the pellet is detached from the tube wall.

7.      After centrifugation (10,000 g x 5 minutes), the resultant supernatant is drained.

8.      Add 1500µL of Washing Solution (B) containing glycogen to the tube and vortex. After centrifugation (10,000 g x 5 minutes), discard the supernatant. The resulting pellet contains DNA and carrier glycogen.

9.      Reconstitute the pellet in Zero Calibrator.

Technical Note

If the sample contains DNAase then step 2 follows step 3. When sample is insoluble or falls out of solution during refrigeration, increase incubation temperature to 40-60ºC. Sample of high concentration, however, may cause aggregation.

References:

Ishizawa, M., Kobayashi, T., and Matsura, S., "Simple Procedure of DNA Isolation from Human Serum," Nucleic Acids Res., 19 (1991) 5792.

Workman, Wesley E., "Sample Preparation and Residual DNA Analysis of Biopharmaceuticals," Pharmacopeial Forum, 21 (March-April 1995) 479-484.

*Note:     Light addressable potentiometric sensor (LAPS) system for the quantitation of DNA and proteins in biopharmaceuticals. Produced by Molecular Devices Corp. (Sunnyvale, CA).