Experiment-6

ISOLATION OF DNA FROM HUMAN BLOOD

15BBT0235

Kannan C

Aim:

Isolation of total DNA from human whole blood sample.

Principle:

This extraction protocol avoids using phenol-chloroform; instead, high salt concentration is used
to remove proteins. Hence the method is quick, safe and cost effective. Tris HCl functions as a
buffering agent, Sodium chloride lyses the cells, so that the DNA is stabilized and it is
maintained in its duplex form. EDTA chelates Mg++ ions which are a cofactor of DNase,
presence of DNase degrades the DNA rapidly. Anionic detergent SDS disrupts the lipid bi-layers
which helps to dissolve the cell membrane and binds the positively charged proteins to release
the DNA into the solution. Sodium perchlorate acts as deproteinizing agent and ethanol performs
the role of a precipitating agent.

Protocol:

1) Collect 1.5 ml of blood in a 15 ml sterile falcon tube and add 6 ml of Reagent A.

2) Mix it in a shaker for 5 minutes at room temperature and centrifuge at 15,000 rpm for 5
minutes at 4C

3) Discard the supernatant; remove the moisture completely by inverting the tube over a tissue
paper and air dry it.

4) Resuspend the pellet in 1 ml of Reagent B.

5) Add 250 l of 5 M sodium per chlorate and mix it by tapping the tubes.

6) Incubate the tubes at 65C for 20 minutes in a water bath and then allow it cool to room
temperature.

7) Add 2ml of ice cold chloroform and mix it for 20 minutes using a shaker.

8) Centrifuge at 10000 rpm for 5 minutes.

9) Transfer the aqueous phase into a new eppendorf tube and add twice the volume of ice cold
absolute alcohol and invert the tube several times gently, as DNA appears as pale threads.
10) Centrifuge at 10000 rpm for 10 minutes and air dry the pellet.

11) Resuspend the pellet in 150 l of TE buffer and resolve it in 0.6 % agarose gel.

Discussion

0.6% agarose gel was used for isolating human DNA.

DNA extraction from the blood cells happens utilizing gel electrophoresis.

Questions:

1. How the DNA extraction from blood cells is different that bacterial cells?

2. What % of Agarose gel is used for separating human DNA and why?

3. What is the role of sucrose in reagent A?

4. How the histone proteins are removed from the DNA while isolating?

Answers

1) How the DNA extraction from blood cells is different that bacterial cells?
DNA extraction from the blood cells happens utilizing gel
electrophoresis while DNA extraction from bacterial cells is done utilizing
centrifugation.
2) What % of Agarose gel is used for separating human DNA and why?
0.6% Agarose gel is utilized for isolating human DNA to distinguish its
atomic size and trustworthiness and to generally estimate the DNA content.
3) What is the role of sucrose in reagent A?
Sucrose stabilizes lysosomal membranes.
4) How the histone proteins are removed from the DNA while isolating?
Histones are removed by proteinase K digestion and/or phenol chloroform
extraction.