Cell Bio Lab 6: Genomic DNA Extraction

0.0(0)
Studied by 0 people
call kaiCall Kai
learnLearn
examPractice Test
spaced repetitionSpaced Repetition
heart puzzleMatch
flashcardsFlashcards
GameKnowt Play
Card Sorting

1/10

encourage image

There's no tags or description

Looks like no tags are added yet.

11 Terms

1
New cards

What are the 6 steps of Genomic DNA extraction from adult Drosophila?

  1. Tissue homogenization

  2. Cell lysis in DNA extraction buffer

  3. Separation of DNA from proteins & other cellular components

  4. DNA precipitation

  5. DNA washing

  6. DNA resuspension for downstream experiments


2
New cards

What does homogenization do?

It breaks apart the flies and their tissues, allowing the extraction buffer to reach the cells

  • Note: Drosophila cells DON’T have a cell wall


3
New cards

What does the genomic DNA extraction buffers used in this experiment have in them?

  • Tris-HCl

  • EDTA

  • SDS


4
New cards

What does Tri-HCl do?

  • Maintains a stable pH during extraction

  • Helps protect DNA


5
New cards

What does EDTA do?

Binds divalent cations (i.e. Mg2+ and Ca2+) → reduces activity of DNases that require these ions → helps prevent DNA degradation


6
New cards

What does SDS do?

  • Is a detergent that disrupts cell and nuclear membranes

  • Denatures proteins, releasing DNA into solution


7
New cards

What incubation temperature promotes cell lysis and protein denaturation?

70°C

8
New cards

What is done after lysis?

  • 1) Potassium acetate is added to precipitate SDS along w/ associated proteins, lipids, and cellular debris

  • 2) Centrifugation then collects these contaminants in a pellet, while the DNA remains in the supernatant

  • 3) The supernatant is then transferred to a clean tube, and isopropanol is added to reduce DNA solubility and precipitate it

  • 4) The DNA is collected by centrifugation and washed w/ 70% ethanol to remove residual salts & other soluble contaminants

  • 5) After ethanol evaporates, DNA pellet is resuspended in TE buffer or water for concentration measurement & downstream experiments such as PCR


9
New cards

How can you assess the concentration & purity of extracted genomic DNA?

By using a NanoDrop spectrophotometer

10
New cards

At what absorbance does DNA absorb ultraviolet light? What is its absorbance used for?

  • 260 nm

  • Its absorbance is used to estimate concentration


11
New cards

What A260/A280 ratio is typical of pure DNA?

  • 1.8 and 2.0 - 2.2

  • Lower ratios = indicate protein contamination or residual salts/other extraction reagents

  • Higher ratios = reflect RNA contamination or measurement conditions

  • Note: these ratios assess purity but DON’T measure DNA integrity → DNA integrity can be examined by agarose gel electrophoresis