Pr3 Flashcards
(24 cards)
What is the purpose of DNA isolation from blood?
It is a critical procedure in molecular biology.
What role does SDS (Sodium Dodecyl Sulfate) play in DNA isolation?
It is a detergent used to lyse cells and release DNA.
What is the preferred anticoagulant for blood samples in DNA isolation?
EDTA.
List three materials needed for DNA isolation from blood.
- Fresh or frozen blood sample
- SDS
- Proteinase K
What is the first step in the DNA isolation protocol?
Lysis of Red Blood Cells.
What is the centrifuge speed and duration for pelleting white blood cells?
3,000 rpm for 5 minutes.
What is added to the pellet to disrupt white blood cells?
Nuclei Lysis Buffer.
What is the final concentration of Proteinase K added during white blood cell lysis?
100-200 μg/ml.
Fill in the blank: After adding Phenol:Chloroform solution, the solution is mixed gently by _______.
inverting the tube.
What should be done to the DNA after precipitation?
Wash the pellet with 70% ethanol.
How should the DNA pellet be resuspended?
In DNase-free water or TE buffer.
What is the ideal 260/280 nm ratio for measuring DNA concentration and purity?
1.8-2.0.
What precautions should be taken when handling phenol and chloroform?
Handle in a fume hood due to toxicity.
What is a common application for the extracted DNA?
PCR, sequencing, electrophoresis, and molecular diagnostics.
What are the components of the lysis buffer for red blood cell lysis?
- 10 mM Tris-HCl (pH 7.5)
- 10 mM NaCl
- 25 mM EDTA
- 0.5% SDS
What is the storage condition for the Red Blood Cell Lysis Buffer?
Store at 4°C for up to 6 months.
What is the pH of the Nuclei Lysis Buffer?
8.0.
What is the purpose of TE buffer in molecular biology?
To preserve DNA or RNA.
List the components of TE Buffer.
- 10 mM Tris-HCl (pH 8.0)
- 1 mM EDTA (pH 8.0)
What is the molecular weight of Tris-HCl used in TE Buffer preparation?
121.14 g/mol.
How much EDTA is needed for 1 liter of TE Buffer?
0.2922 g.
What should be done to the solutions and tools to preserve DNA integrity?
Ensure they are free of DNase contamination.
What is the method of sterilization for preparing lysis buffers?
Autoclaving or filtering through a 0.22 µm filter.
True or False: Ethanol can be used instead of isopropanol for DNA precipitation.
True.
