Download MBBS Methods for the Isolation of DNA Lecture PPT

Download MBBS (Bachelor of Medicine and Bachelor of Surgery) Latest Methods for the Isolation of DNA Lecture PPT

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

Phenol/chloroform extraction method

Silica gel extraction method

Proteinase K extraction method

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

Required quantity

Purity of DNA

Time

Molecular weight of DNA
Equipment/reagent requirements

Red blood cell lysis buffer
White blood cell lysis buffer
RNAase
Protein precipitation solution
Isopropanol
DNA hydration buffer
A centrifuge machine
A vortex mixer
-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

(EDTA tube) or a cell pellet following plasma

separation from blood collected in EDTA

tube

Specimen can be stored at 4 degree Celsius

for 48 hours prior to processing.


Procedure

Cell lysis

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

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.

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

cell pellet and vortex vigorously for 10 sec.

4. RNAase is added to remove RNA from the preparation

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

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

form a tight,dark brown pellet.

DNA precipitation

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

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

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

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

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

degree C for 1 hour to dissolve DNA.

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

purity as well as to known the approximate

concentration of DNA