Pr1 Flashcards
(57 cards)
What is the purpose of DNA isolation?
To extract DNA from cells for applications such as cloning, sequencing, and diagnostics.
What are the basic steps in DNA isolation?
- Cell Lysis
- Removal of Proteins and Other Contaminants
- Precipitation of DNA
- Rehydration of DNA
What is the principle behind the Organic Extraction method?
Uses organic solvents such as phenol and chloroform to separate DNA from proteins and other contaminants.
List the steps involved in the Organic Extraction method.
- Lyse the cells using a lysis buffer containing SDS
- Add equal volumes of phenol-chloroform and centrifuge
- DNA remains in the aqueous phase
- Precipitate DNA with ethanol or isopropanol
What is an advantage of the Organic Extraction method?
High purity DNA suitable for sensitive downstream applications.
What is a disadvantage of the Organic Extraction method?
Time-consuming and uses toxic chemicals.
How does the Salting-Out Method work?
Relies on high salt concentrations to precipitate proteins, leaving DNA in solution.
List the steps involved in the Salting-Out Method.
- Lyse cells using a simple lysis buffer
- Add a high-salt solution to precipitate proteins
- Centrifuge to remove protein pellet
- Precipitate DNA with alcohol
What is an advantage of the Salting-Out Method?
Non-toxic and inexpensive.
What is a disadvantage of the Salting-Out Method?
May result in lower purity compared to organic extraction.
What principle does the Silica Column-Based Method utilize?
DNA binds to silica in the presence of chaotropic salts.
List the steps involved in the Silica Column-Based Method.
- Lyse cells using a chaotropic salt-containing buffer
- Pass the lysate through a silica column
- Wash away contaminants
- Elute purified DNA with a low-salt buffer or water
What is an advantage of the Silica Column-Based Method?
Quick and efficient; no hazardous chemicals.
What is a disadvantage of the Silica Column-Based Method?
Relatively expensive due to the need for commercial kits.
What principle does the Magnetic Bead-Based Method operate on?
Magnetic beads coated with DNA-binding materials capture DNA from a lysate.
List the steps involved in the Magnetic Bead-Based Method.
- Lyse cells and add magnetic beads to the lysate
- Use a magnet to separate the beads and bound DNA
- Wash the beads and elute the DNA
What is an advantage of the Magnetic Bead-Based Method?
Automatable and scalable for high-throughput.
What is a disadvantage of the Magnetic Bead-Based Method?
Costly compared to traditional methods.
What is the principle behind the Chelex® Resin Method?
Chelex resin binds metal ions, inactivating nucleases that could degrade DNA.
List the steps involved in the Chelex® Resin Method.
- Boil the sample with Chelex resin
- Centrifuge to separate DNA-containing supernatant
What is an advantage of the Chelex® Resin Method?
Simple and quick; minimizes DNA degradation.
What is a disadvantage of the Chelex® Resin Method?
Limited to small-scale applications and PCR-ready DNA.
What is the Alkaline Lysis method used for?
Isolating plasmid DNA from bacterial cells.
What does the CTAB Method specifically target?
Polysaccharides and secondary metabolites commonly found in plants.
