Lab practical 5

Aim:

Isolation and purification of genomic DNA from whole blood (using spin columns).

Introduction:

Blood is a specialized body fluid composed of cells suspended in a liquid called blood plasma. Whole blood
contains three types of cells:

1. Red blood cells (RBCs)

2. White blood cells (WBCs)
3. Platelets

Red blood cells (RBCs) do not have any DNA, as they lose their nuclei during maturation. The white blood cell
(WBC) component of the blood contains DNA. The blood sample is treated with detergents which break
open the cell membrane to release the contents. Enzymes are then used to break down all the proteins, RNA,
sugars and fats in the solution.
Blood Drop White Blood Cells (WBCs) Double Stranded DNA

Fig 1: Extraction of blood genomic DNA from white blood cells (WBCs)

Blood Genomic DNA Extraction teaching Kit (Column Based) is designed for rapid extraction and purification of
pure genomic DNA from whole blood. It has many advantages over the crude methods of DNA extraction
and does not contain harmful organic compounds such as phenol and chloroform.

The DNA purification procedure using the miniprep spin columns comprises of three steps:
 Adsorption of DNA to the membrane
 Removal of residual contaminants
 Elution of pure genomic DNA
The columns have a high binding capacity and high quality DNA can be obtained from various species.

Principle:

Blood Genomic DNA Extraction Teaching Kit (Column Based) simplifies the isolation of DNA from fresh blood with
spin column procedure. Genomic DNA purification from blood involves cell lysis which is achieved by incubation of
whole blood in a solution containing chaotropic ions in the presence of Proteinase K at 55oC which helps in
the digestion of tissue and cell membranes. It is then applied onto Miniprep Spin Column which contains specially
developed membranes for optimal binding of genomic DNA. After the initial binding of DNA, impurities like
proteins,
polysaccharides, low molecular weight metabolites and salts are removed by short washing steps. High quality
DNA is finally eluted in the Elution Buffer.

Kit Contents:
The kit can be used to extract genomic DNA from whole blood.

Table 1: Enlists the materials provided in this kit with their quantity and recommended storage

Sr. Quantity
Product
No. Materials Provided Storage
Code 10 expts 20 expts
1 TKC009 Control DNA 0.11 ml 0.22 ml -20oC
2 DS0010 Lysis Solution 2.5 ml 5 ml RT
3 DS0011 Prewash Solution 6 ml 11.5 ml RT
4 DS0012 Wash Solution 6 ml 11.5 ml RT
5 DS0040 Elution Buffer 2.5 ml 5 ml RT
6 DS0013 Proteinase K Solution 0.25 ml 0.5 ml -20oC
7 DS0003 RNase A Solution 0.25 ml 0.5 ml RT
11 DBCA02 Miniprep Spin Column (Collection Tube) 11 Nos. 22 Nos. RT

12 PW1139 Collection Tubes, Polypropylene (2.0 ml) 22 Nos. 44 Nos. RT

Storage:

Blood Genomic DNA Extraction Teaching Kit (Column Based) is stable for 6 months from the date of receipt
without showing any reduction in performance. On receipt, store the Control DNA and Proteinase K at -20oC. 6X
Gel Loading Buffer should be stored at 2-8 oC. Other kit contents can be stored at room temperature (15-
25oC).

Important Instructions:

1. Preheat a water bath or heating block to 55oC.

2. Thoroughly mix the reagents. Examine the reagents for precipitation. If any kit reagent forms a
precipitate (other than enzymes), warm at 55-65oC until the precipitate dissolves and allow it to
o
cool down to room temperature (15-25 C) before use.
3. Ensure the use of only clean & dry eppendorf tubes and tips for the procedure.
4. Ensure that the blood is collected under sterile conditions in an anticoagulant coated tube (e.g.
EDTA).
Procedure:

Read the Important Instructions before starting the experiment.

1. Take 200 µl of the fresh whole blood in a 2.0 ml collection tube. Ensure that the
blood sample is at room temperature (15-25oC) before beginning the protocol.
2. Add 20 µl of the Proteinase K solution into the above collection tube containing
blood. Vortex (10-15 seconds) to ensure thorough mixing of the enzyme.

3. Add 20 µl of RNase A solution. Vortex (10-15 seconds) to ensure thorough mixing

of the enzyme and incubate for 2 minutes at room temperature.

NOTE: This step helps in getting RNA-free genomic DNA.

4. Lysis Reaction
Add 200 µl of the Lysis Solution to the sample, vortex thoroughly for a few seconds to
obtain a homogenous mixture. Incubate at 55oC for 10 minutes.

NOTE: If cell clumps are visible, pipette the sample gently to obtain a homogenous
mixture.

5. Prepare for Binding

Add 200 µl of ethanol to the lysate obtained from step 4 and mix thoroughly by
gentle pipetting for 5-10 seconds.

NOTE: A homogenous solution should be obtained after addition of ethanol.

6. Load lysate in HiElute Miniprep Spin Ccolumn

Transfer the entire lysate obtained from step 5 into HiElute Miniprep Spin Column.
Centrifuge at 10,000 rpm for 1 minute. Discard the flow-through liquid and place the
column in a new 2.0 ml collection tube.

NOTE: Use a wide bore pipette tip to reduce shearing of the DNA when transferring
contents onto the column.

7. Prewash
Add 500 µl of Prewash Solution to the HiElute Miniprep Spin Column and
centrifuge at 10,000 rpm for 1 minute. Discard the flow-through liquid and re-use
the same collection tube with the column.

8. Wash
Add 500 µl of Wash Solution to the column and centrifuge at 14,000 rpm for 3
minutes. Discard the flow through. Place the column in the same collection tube and
centrifuge it for an additional one minute at 14,000 rpm to remove traces of Wash
Solution. Discard the collection tube and place the column in a new 2.0 ml
collection tube.

9. DNA Elution
Add 200 µl of the Elution Buffer directly into the column without spilling to the
o
sides. Incubate for 1 minute at room temperature (15-25 C). Centrifuge at 10,000
rpm for 1 minute to elute the DNA.

Storage of the eluate with purified DNA: The eluate contains pure genomic DNA. For
short term storage (24- 48 hours) of the DNA, 2-8oC is recommended. For long-term
storage, -20oC or lower temperature (-80oC) is recommended. Avoid repeated freezing
and thawing of the sample which may cause denaturing of DNA. The Elution Buffer
will help to stabilize the DNA at these temperatures.