LAB PRACTICAL 3

TITLE: BLOOD DNA EXTRACTION

OBJECTIVE: To extract DNA of interest from blood by using commercially available DNA extraction kit.

INTRODUCTION:

DNA extraction is routine procedure to obtain DNA for subsequent molecular application such as restriction enzyme digestion, polymerase chain reaction (PCR), cloning and et cetera. DNA extraction consists of 3 basic steps in order to make sure sufficient amount of DNA will be extracted. The first step is to lyse the cells containing the DNA of interest. The second step is removing the lipid membrane by adding detergent and the last step is to precipitate DNA with alcohol.

MATERIALS:

Genomic DNA Mini Kit

1. GD columns

2. Collection tubes

3. RBC Lysis Buffer

4. GB Buffer

5. W1 buffer (ethanol added)

6. 1.5 mL microcentrifuge tube

PROCEDURES

Genomic DNA Mini Kit is designed for rapid and efficient purification of genomic DNA from blood without the need for precipitation or organic extraction. This kit uses a specially-treated glass filter membrane fixed into a column to efficiently bind DNA in the presence of high salt. The kit applies the principle of minicolumn spin technology and the use of optimized buffers to ensure that only DNA is isolated while proteins and other impurities, additives, preservatives are removed during the subsequent washing steps. High purify genomic DNA is then eluted in water or low salt buffers has a A260/280 ratio between 1.7 and 1.9, making it ready to use in many routine molecular biology application such as restriction enzyme digestion, Southern blotting, DNA fingerprinting, PCR and other manipulation.

1. Pre set waterbath to 60oC.

2. Sample Preparation

Transfer ~300uL of blood to a 1.5mL microcentrifuge tube.

Add 3X the sample volume or RBC Lysis Buffer, mix by inversion

Incubate the tube, 10 mins at room temperature.

Centrifuge for 5 mins at 3000xg, then remove the supernatant.

Add 100uL of RBC Lysis Buffer to resuspend the pellet.

3. Cell lysis

Add 200uL of GB Buffer to the 1.5mL microcentrifuge tube, then shake vigorously.

Incubate at 60oC for 10 mins to ensure lysate is clear.

During incubation, invert every 3 mins

At this time preheat the required Elution Buffer (200 uL per sample) to 60oC. (for DNA Elution)4. DNA binding

Add 200uL of absolute ethanol to the lysate then immediately mix by shaking vigorously for 10 secs.

Note: If precipitate appears, break it up as much as possible with pipet.

Place a GD column in a 2mL Collection tube.

Centrifuged at 14,000-16,000 x g for 5 minutes

Discard the 2mL Collection tube then place the GD column in a new 2mL Collection tube.

5. Wash

Add 400uL of W1 Buffer to the GD column then centrifuge at 14-16,000xg for 30-60 secs.

Discard the flow-through then place the GD column back in the 2mL collection tube.

Add 600 uL of Wash Buffer (added ethanol) to the GD column.

Centrifuge at 14-16,000xg for 30-60 secs. Then discard the flow-through.

Place the GD column back in the 2mL Collection tube.

Centrifuge again for 3 mins at 14-16,000xg to dry the column matrix.

6. DNA elution

Transfer the dried GD column to a clean 1.5mL microcentrifuge tube.

Add 100uL of pre-heated Elution Buffer to the centre of column matrix.

Lets stand for at least 3 mins to ensure the Elution Buffer is completely absorbed.

Centrifuge at 14-16,000xg for 30 secs to elute the purified DNA.

Store DNA at 4oC or 20oC.

QUESTIONS:

1. List the methods that can be used to lyse a cell.

2. Why does DNA can precipitate? (in Step 4)

3. State the function of the following:

(a) Lysis buffer

(b) Ethanol

(c) Detergent

4. How long will DNA last? Will it eventually degrade or disappear?