S012617 // Josh Sharp
Unit 13 : BC
DNA Analysis, Extraction and Amplification
DNA Extraction
DNA Extraction is a process that allows scientists to derive pure DNA
material from a substance, such as hair, bodily fluids or skin cells. It
separates genetic material from other intracellular structures and proteins
to give clear results later when undergoing DNA testing. It includes
breaking down the cell into individual components, separating the DNA
from proteins and purification to ensure that the end product contains
only DNA. DNA extraction is used commonly in forensics when trying to
identify genetic material, as well as studying and diagnosing genetic
diseases/disorders. If we did not extract DNA, the DNA is still encapsulated
in a membrane and later tests such as PCR and Electrophoresis would not
be as efficient or accurate.
Reagents
- 8 oz Styrofoam Cups
- 2ml Microcentrifuge Tubes
- 15ml Centrifuge Tubes
- Bulb Type Transfer Pipettes
- Bio-Rad Chelex-100 Molecular Biology Grade Resin
- HPLC Grade Water
- Microcentrifuge
- Water Bath or Heat Block
Method
1. Place 10Ml of HPLC Grade water in the 8oz Styrofoam cup using a
sterile pipette.
2. Place the water in your mouth and swish vigorously for 45 seconds.
This will gather cheek cells from the lining in your mouth. Spit the
water back into the cup with care and carefully pour contents into a
15ml tube.
3. Cap the tube and leave it to stand for 15 minutes, or until the solid
cells set at the base of the tube.
4. Gather the solid cells from the bottom of the tube using a bulb
pipette, taking care to minimise the quantity of liquid you collect.
5. Place the collected cells in a 2ml microcentrifuge and place in the
centrifuge at maximum speed for 5 minutes to produce a pellet at
the base of the tube.
pg. 1
, S012617 // Josh Sharp
Unit 13 : BC
6. Pour out as much liquid as possible without losing pellet or genetic
information.
7. Add 100µL of Chelex Resin to the tube.
8. Incubate the suspension 100 degrees Celsius for 10 minutes, the heat will break the
bonds in the DNA and release the proteins.
9. Place the tubes into ice for 3 minutes. This will allow the Chelex resin to bind with
everything but the DNA.
10. Centrifuge the tube for 5 minutes at maximum speed. The Chelex bound
material and pellet will drop to the bottom of the tube whilst the liquid above will
contain pure DNA only.
11. Transfer liquid only to clear small tube, resulting in pure DNA.
Solid Phase Extraction
Reagents
- 8oz Styrofoam Cups
- 2ml Microcentrifuge Tubes
- 15ml Centrifuge Tubes
- Bulb Type Transfer Pipette
- Syringe
- pH10 buffer
- Acetonitrile
- Ammonium Acetate
- Nuclear Lysis Solution (Detergent)
- Alcohol
- SPE Tube
- Low Salt Solution
- Waste Beaker
Method
1. Place 10Ml of HPLC Grade water in the 8oz Styrofoam cup using a
sterile pipette.
2. Place the water in your mouth and swish vigorously for 45 seconds.
This will gather cheek cells from the lining in your mouth. Spit the
water back into the cup with care and carefully pour contents into a
15ml tube.
3. Add one small drop of Nuclear Lysis Solution to aid in the breakdown
of the DNA bonds.
4. Prepare the extraction column (drip rate medium)
5. Add 3ml pH 10 buffer to centrifuge tube
6. Add 0.5ml acetonitrile with a syringe for accuracy into centrifuge
tube
pg. 2
Unit 13 : BC
DNA Analysis, Extraction and Amplification
DNA Extraction
DNA Extraction is a process that allows scientists to derive pure DNA
material from a substance, such as hair, bodily fluids or skin cells. It
separates genetic material from other intracellular structures and proteins
to give clear results later when undergoing DNA testing. It includes
breaking down the cell into individual components, separating the DNA
from proteins and purification to ensure that the end product contains
only DNA. DNA extraction is used commonly in forensics when trying to
identify genetic material, as well as studying and diagnosing genetic
diseases/disorders. If we did not extract DNA, the DNA is still encapsulated
in a membrane and later tests such as PCR and Electrophoresis would not
be as efficient or accurate.
Reagents
- 8 oz Styrofoam Cups
- 2ml Microcentrifuge Tubes
- 15ml Centrifuge Tubes
- Bulb Type Transfer Pipettes
- Bio-Rad Chelex-100 Molecular Biology Grade Resin
- HPLC Grade Water
- Microcentrifuge
- Water Bath or Heat Block
Method
1. Place 10Ml of HPLC Grade water in the 8oz Styrofoam cup using a
sterile pipette.
2. Place the water in your mouth and swish vigorously for 45 seconds.
This will gather cheek cells from the lining in your mouth. Spit the
water back into the cup with care and carefully pour contents into a
15ml tube.
3. Cap the tube and leave it to stand for 15 minutes, or until the solid
cells set at the base of the tube.
4. Gather the solid cells from the bottom of the tube using a bulb
pipette, taking care to minimise the quantity of liquid you collect.
5. Place the collected cells in a 2ml microcentrifuge and place in the
centrifuge at maximum speed for 5 minutes to produce a pellet at
the base of the tube.
pg. 1
, S012617 // Josh Sharp
Unit 13 : BC
6. Pour out as much liquid as possible without losing pellet or genetic
information.
7. Add 100µL of Chelex Resin to the tube.
8. Incubate the suspension 100 degrees Celsius for 10 minutes, the heat will break the
bonds in the DNA and release the proteins.
9. Place the tubes into ice for 3 minutes. This will allow the Chelex resin to bind with
everything but the DNA.
10. Centrifuge the tube for 5 minutes at maximum speed. The Chelex bound
material and pellet will drop to the bottom of the tube whilst the liquid above will
contain pure DNA only.
11. Transfer liquid only to clear small tube, resulting in pure DNA.
Solid Phase Extraction
Reagents
- 8oz Styrofoam Cups
- 2ml Microcentrifuge Tubes
- 15ml Centrifuge Tubes
- Bulb Type Transfer Pipette
- Syringe
- pH10 buffer
- Acetonitrile
- Ammonium Acetate
- Nuclear Lysis Solution (Detergent)
- Alcohol
- SPE Tube
- Low Salt Solution
- Waste Beaker
Method
1. Place 10Ml of HPLC Grade water in the 8oz Styrofoam cup using a
sterile pipette.
2. Place the water in your mouth and swish vigorously for 45 seconds.
This will gather cheek cells from the lining in your mouth. Spit the
water back into the cup with care and carefully pour contents into a
15ml tube.
3. Add one small drop of Nuclear Lysis Solution to aid in the breakdown
of the DNA bonds.
4. Prepare the extraction column (drip rate medium)
5. Add 3ml pH 10 buffer to centrifuge tube
6. Add 0.5ml acetonitrile with a syringe for accuracy into centrifuge
tube
pg. 2
