Download MBBS Methods for the Isolation of DNA Lecture PPT

--- Content provided by⁠ FirstRanker.com ---

Silica gel extraction method

Proteinase K extraction method

--- Content provided by‌ FirstRanker.com ---

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

Required quantity

--- Content provided by‌ FirstRanker.com ---

Purity of DNA

Time

Molecular weight of DNA

--- Content provided by​ FirstRanker.com ---

Equipment/reagent requirements

Red blood cell lysis buffer
White blood cell lysis buffer
RNAase

--- Content provided by‍ FirstRanker.com ---

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

--- Content provided by‌ FirstRanker.com ---

-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

--- Content provided by⁠ FirstRanker.com ---

(EDTA tube) or a cell pellet following plasma

separation from blood collected in EDTA

--- Content provided by FirstRanker.com ---

tube

Specimen can be stored at 4 degree Celsius

for 48 hours prior to processing.

--- Content provided by FirstRanker.com ---

Procedure

Cell lysis

--- Content provided by‍ FirstRanker.com ---

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

--- Content provided by‌ FirstRanker.com ---

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.

--- Content provided by⁠ FirstRanker.com ---

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

cell pellet and vortex vigorously for 10 sec.

--- Content provided by‌ FirstRanker.com ---

4. RNAase is added to remove RNA from the preparation

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

--- Content provided by​ FirstRanker.com ---

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

--- Content provided by​ FirstRanker.com ---

form a tight,dark brown pellet.

DNA precipitation

--- Content provided by‌ FirstRanker.com ---

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

--- Content provided by‍ FirstRanker.com ---

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

--- Content provided by‍ FirstRanker.com ---

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

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

--- Content provided by‍ FirstRanker.com ---

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

degree C for 1 hour to dissolve DNA.

--- Content provided by‌ FirstRanker.com ---

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

--- Content provided by‍ FirstRanker.com ---

purity as well as to known the approximate

concentration of DNA