Experiment 3: DNA extraction from blood

Preparation of Reagents

a.TKM-1 Buffer / Low salt buffer(500 ml): 0.605 g of TrisHCl (10mM) pH 7.6, 0.372 g of
KCl (10 mM), 1.016 g of MgCl2(10 mM), 0.372g of EDTA (2mM) was dissolved in 500ml of
distilled water.

b. Triton-X (10ml): Added 0.1 ml of 100 % Triton-X to 9.9ml of distilled water.

c.TKM-2 Buffer / High salt buffer (100 ml): 0.121 g of TrisHCl (10mM) pH 7.6, 0.074 g
ofKCl (10 mM), 1.203 g of MgCl2(10 mM), 0.074 g EDTA (2mM), 0.467 g of NaCl (0.4 M)
was dissolved in 100ml of distilled water.

d. SDS: One gram of sodium dodecyl sulphate was dissolved in 10ml distilled water.

e. 6M NaCl: 8.765 g of NaCl was dissolved in 25 ml of distilled water

f. TE Buffer: 0.030 g of TrisHCl (10mM) pH 8.0, 0.009 g of EDTA (1mM) was dissolved in
100ml of distilled water

RBC Lysis

1. Take 300 µl of EDTA blood in an autoclaved 1.5 ml eppendorf tube.

2. Add 900 µl of TKM 1 and 50 µl of 1x Triton-X.

3. Incubated at 370C for 5 minutes to lyses the RBCs.

4. Centrifuge at 8000 rpm for 3 minute and discard the supernatant.

5. Repeat this step 2-3 times with decreasing amount of 1x Triton-X till, until complete lyses of
RBC and a white pellet of WBCs remaining.

Biotechnology Department 2018

Cell Lysis

6. Add 300 µl of TKM 2 and 40 µl of 10% SDS to the cell pellet, Mix it thoroughly and
incubate at 370 C for 5 minutes.

7. At the end of incubation, add 100 µl of 6M NaCl and vortex it to precipitate the proteins.

8. Centrifuge at 8000 rpm for 5 minutes.

Precipitation of DNA

9. Transfer the supernatant in to a new eppendorf tube.

10. Add 300 µl of isopropanol and inverting the eppendorf slowly to facilitate precipitation.

11. Centrifuge the eppendorfs at 8000 rpm for 10 minutes to pellet down the DNA.

12. Discard the supernatant; add 70% ethanol and mix slowly to remove any excess salts.

13. Centrifuge at 8000 rpm for 5 minutes to pellet down the DNA.

14. Discard the supernatant and air dry the DNA.

15. After thorough drying, 50 µl of TE buffer will be added to dissolve the DNA and store it -20
o
c for the next work.

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TKM1

Tris HCl is used to maintain the right pH for DNA isolation. It also interacts with the
lipopolysaccharides present on the outer membrane which helps to permeabilize the membrane.
EDTA is added to accelerate this effect.

EDTA act as cation chelating agent. hence it is used in DNA isolation to chelate the Mg++ ions
which are necessary in activity of nucleases so your DNA will be safe in presence of EDTA. but
higher concentration of EDTA might hamper the further reaction since it should be optimized.
Tris-HCL act as stabilizing buffer during isolation of DNA, which maintain the pH of buffer.

KCl the pellet formed will contain the polysaccharides and non digested tissue. The supernatant
is extracted from the tube and used in the next steps of the DNA extraction.

MgCl2 When membranes are busted by TRIS, there is no compartmentalization in the solution
anymore. MgCl2 is then used because it binds to DNA and thus protects it against DNase
proteins that are now (because of lack of membranes) in direct contact with your DNA.

The binding of MgCl2 to DNA denies access of DNase to the DNA, and your DNA will not be
broken down.

MgCl2 is used to preserve the integrity of membrane system by counteracting the fixed negative
charges of membrane phospholipid

Triton X-100 is widely used to lyse cells to extract protein or organelles, or to permeabilize the
membranes of living cells.

Biotechnology Department 2018

Biotechnology Department 2018