DNA Extraction

Question 1

5 Steps of DNA Extraction

Answer 1

1. Tissue Disruption (grinding and pulverisation)
2. Cell disruption/lysis (SDS detergent, guanidinium salt)
3. Remove other cellular components; mainly proteins. (proteinase K, heat, SDS)
4. Separation of DNA from lysate (organic extraction, salting out, binding to column)
5. Isolate the DNA and purify it (absolute ethanol/isopropanol precipitation + salt)

Question 2

What does EDTA do?

Answer 2

1. Chelating agent for ions like Mg
2. Inactivates nucleases

Question 3

Organic Extraction (phenol and chloroform extraction)

Answer 3

• Add sample to cell lysis buffer
• Remove swab and add phenol
• Vortex and centrifuge
• 3 Layers formed
  Top layer= DNA
  Middle= Denatured proteins
  Bottom= Organic layer (phenol)

Question 4

Advantages of Organic Extraction

Answer 4

• Large quantity of pure and high molecular DNA

Question 5

Disadvantages of Organic Extraction

Answer 5

• Hazardous- toxic phenol
• Time consuming
• Multiple tubes- contamination

Question 6

Chelex Resin

Answer 6

• Ion exchange resin that chelates metal ions (Mg)
• Swab in lysis buffer
• Remove and centrifuge
• Add chalet solution
• Boil @ 100 degrees
• Centrifuge to pellet resin
• DNA ready in supernatant.

Question 7

Advantages of Chelex Resin.

Answer 7

• Quick and cheap
• Single tube
• Non-Hazardous
• Suitable for small samples

Question 8

Disadvantages of Chelex Resin

Answer 8

• Crude
• Can result in inhibition of PCR if too much sample is used.

Question 9

FTA Paper

Answer 9

• absorbent paper that contains chemicals that lyse cells, denature proteins and protects nucleic acids.
• spot cells/blood onto paper
• wash in solvent to remove harm and other potential PCR inhibitors
• rinse in buffer
• use card for PCR

Question 10

Advantages of FTA Paper

Answer 10

• DNA stored on card is stable at room temperature for years
• Quick and sample
• Non Hazardous
• Extract process can be automated

Question 11

Disadvantages of FTA Paper

Answer 11

• Dry paper pieces jump out of PCR tube due to static electricity.

Question 12

Quiagen Kits

Answer 12

• Cell lysis using guanidinium and proteinase K digestion.
• Binding of DNA to silica membrane in column
• Washing of membrane to remove proteins and other contaminants
• Pure DNA is eluted from the membrane.

Question 13

Advantages of Quiagen Kits

Answer 13

• Rapid and consistent
• High quality and yield of DNA
• Complete removal of contaminants

Question 14

Disadvantages of Quiagen Kits

Answer 14

• Expensive

Question 15

Puregene

Answer 15

• Cell lysis in buffer containing TRIS, EDTA and SDS.
• Proteins precipitated out using high concentration of ammonium acetate.
• DNA concentrated (precipitated out) using isopropanol at -20 degrees.
• DNA washed with 70% ethanol to wash the salts and dissolved in buffer.

Question 16

Advantages of Puregene

Answer 16

• Rapid
• Consistent
• Inexpensive
• Non-Hazardous