DNA Extraction & Micropippette

model organism

a non-human species that is used in laboratory to help scientists understand biological processes

A way to distinguish sex among flies

Presence of epandium (males)

Presence of ovipositor (females)

Micropipettes

dispense small volumes of liquids

What is Micropipette labeled as?

• Volumes in uL

• usually 1/10th max in volume

• Types: P2, P10, P20, P100, P200, P1000

DNA Extraction

A process to isolate DNA from cells and proteins

1. DNA extraction Step 1: Cell Lysis

breaks up the cells in DNA

Components of Lysis Buffer during DNA extraction

• SDS (sodium dodecyl sulfate)

• EDTA (metal chelation)

• NaCl+ (positively-charged salt)

• Tris-HCl (pH 8.0)

SDS

Detergent. Disrupts the cell membranes & denatures proteins

EDTA (Mg2+)

Inhibits DNA-degrading enzymes such as DNase

NaCl( positively charged salt)

Creates a protective layer around the negatively charged DNA (blue)

Tris-HCl

a pH buffer to make it pH (8.0)

2. DNA extraction Step 2: Protein Removal

The bulk of the cell protein needs to be removed

• KOAc: concentrated salt solution. K+ binds and precipitates out SDS-bound proteins (Lysis Buffer) and other debris.

• Cold temp = reduce solubility = increase cloudiness

• ALL PROTEIN will be in the pellet

How is separation achieved?

By Centrifuging

• spins samples at 1,000 rpm. Forces the precipitate to form a solid pellet (debris pellet)

• TUBES MUST BE EQUALLY SPACED

• After that, the supernatant (containing the DNA and some salt/debris) must be transferred to a fresh tube

Vortex

mixes small vials of liquids in a rapid motion for 10-15 seconds

Spin (mini centrifuge)

spins down PCR tubes for 10-15 seconds

3. DNA Extraction Step 3: Alcohol precipitation

Isolate DNA from solution containing salt/debris

• Isopropanol: DNA is insoluble in this; it will form a precipitate

• Ethanol: salts/organic molecules are soluble in this; cleans up contaminants from precipitate

4. DNA Extraction Step 4: Rehydrate Pellet

Purified DNA must be rehydrated with a buffered solution

• Why? It will be used for PCR and gel electrophoresis

• DNA is measured in nanograms (ng)