Pr 2 Flashcards
What is the principle of isolating and purifying total DNA from animal blood?
Blood cells are lysed and DNA is precipitated by ethanol, then DNA is separated by adsorbing it onto silicone gel.
List the requirements for DNA isolation from animal blood.
- Micro pipettes
- Vortexer
- Micro centrifuge tubes (1.5 -2 ml)
- Micro centrifuge
- Water bath at 56°C
What is the function of Phosphate buffer saline (PBS) in DNA isolation?
Regulates pH and maintains cell structure.
What is the role of ethanol in the DNA isolation process?
Precipitates the DNA.
What does RNase A do in the DNA isolation procedure?
Degrades the RNA.
What is the purpose of Proteinase K in DNA isolation?
Degrades the proteins.
What is the function of the lysis buffer (AL)?
Breaks the cell membrane.
What does wash buffer AW1 remove?
Removes salts and other molecules from the DNA.
What is the purpose of wash buffer AW2?
Removes ethanol from the DNA.
What is the function of the elution buffer (AE)?
Separates DNA from the silicon membrane.
What are the ingredients for 1 liter of PBS?
- NaCl (Sodium Chloride): 8.0 g
- KCl (Potassium Chloride): 0.2 g
- Na₂HPO₄ (Disodium Phosphate): 1.44 g
- KH₂PO₄ (Monopotassium Phosphate): 0.24 g
- Distilled water (DNase-free): To make up the volume to 1 liter
What pH should PBS be adjusted to?
7.4
How can PBS be sterilized?
- Autoclave at 121°C for 15 minutes
- Filter using a 0.22 µm filter
What type of blood sample is used for DNA isolation?
Freshly drawn blood or frozen blood sample at –20°C or –70°C.
What is the first step in the DNA isolation procedure?
Take a 2 ml microcentrifuge tube and add 20 μl proteinase K, 100 μl anticoagulated blood, and 100 μl PBS.
What is added to the mixture after incubating with RNase A?
200 μl Buffer AL (without ethanol).
At what temperature should the mixture be incubated after adding Buffer AL?
56°C for 10 minutes.
What is the centrifuge speed for the DNA adsorption step?
8000 rpm for 1 minute.
What is done after adding Buffer AW1 during DNA purification?
Centrifuge for 1 minute at 8000 rpm and discard the flow-through.
What is the centrifuge speed for the AW2 wash buffer step?
14,000 rpm for 3 minutes.
What should be done to elute DNA from the silicon membrane?
Add 200 μl Buffer AE directly onto the DNeasy membrane and centrifuge for 1 minute at 8000 rpm.
How many times should the elution step be repeated for more DNA?
Repeat step 7.
