Download MBBS Methods for the Isolation of DNA Lecture PPT

METHODS FOR THE ISOLATION OF DNA
Salting out/salt precipitation method

Phenol/chloroform extraction method

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Silica gel extraction method

Proteinase K extraction method

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Anion exchange methos
The choice of method depends on many factors:

Required quantity

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Purity of DNA

Time

Molecular weight of DNA

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Equipment/reagent requirements

Red blood cell lysis buffer
White blood cell lysis buffer
RNAase

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Protein precipitation solution
Isopropanol
DNA hydration buffer
A centrifuge machine
A vortex mixer

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-80 degree Freezer
Incubator/Water-bath
A spectrophotometer capable of reading at 260 and 280 nm/Nanodrop
Choice of specimen
DNA can be isolated from whole blood

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(EDTA tube) or a cell pellet following plasma

separation from blood collected in EDTA

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tube

Specimen can be stored at 4 degree Celsius

for 48 hours prior to processing.

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Procedure

Cell lysis

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1. Dispense 30ml of red blood cell lysis buffer (NH4CL, NaHCO3,

EDTA) in to a 50 ml centrifuge tube containing 5-10 ml of whole

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blood. Incubate at RT for 5 min, inverting occasionally to mix.

2. Centrifuge the samples at 3,000g for to pellet the white blood

cells. Pour the supernatant to waste.

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3. Add 10ml of white cell lysis buffer (SDS, EDTA) to white blood

cell pellet and vortex vigorously for 10 sec.

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4. RNAase is added to remove RNA from the preparation

.Incubate at 37 degree Celsius for 15 minutes.
Protein Precipitation

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6. Add 3.3ml of ammonium acetate protein precipitation solution, and

vortex vigorously for 20 sec at high speed.

7. Centrifuge for 5 min at 3,000g. The precipitated proteins should

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form a tight,dark brown pellet.

DNA precipitation

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8. Dispense 10ml isopropanol in to a clean 50ml centrifuge tube and

add the supernatant from the previous step.

9.Mix by inverting gently 50 times until DNA is visible as thread or

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clump

10. Centrifuge for 5 min at 3,000g. Carefully discard the supernatant
11. Wash the DNA pellet by adding 10 ml of ethanol and centrifuge for

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5 minutes at 3,000g and remove the supernatant.

DNA Hydration
12. Add 0.3-1.0 ml of DNA hydration buffer and vortex

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for 5 sec at medium speed to mix. Incubate at 65

degree C for 1 hour to dissolve DNA.

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13. Incubate at RT overnight with gentle shaking.

14. The absorbance of the DNA at 260 nm and 280 nm

should be measured using a quartz cuvette to assess

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purity as well as to known the approximate

concentration of DNA