DNA Extraction & Micropippette

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17 Terms

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<p>model organism</p>

model organism

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

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A way to distinguish sex among flies

Presence of epandium (males)

Presence of ovipositor (females)

<p>Presence of epandium (males)</p><p>Presence of ovipositor (females)</p>
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<p>Micropipettes</p>

Micropipettes

dispense small volumes of liquids

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What is Micropipette labeled as?

  • Volumes in uL

  • usually 1/10th max in volume

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

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<p>DNA Extraction</p>

DNA Extraction

A process to isolate DNA from cells and proteins

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<ol><li><p>DNA extraction Step 1: Cell Lysis</p></li></ol><p></p>

  1. DNA extraction Step 1: Cell Lysis

breaks up the cells in DNA

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Components of Lysis Buffer during DNA extraction

  • SDS (sodium dodecyl sulfate)

  • EDTA (metal chelation)

  • NaCl+ (positively-charged salt)

  • Tris-HCl (pH 8.0)

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SDS

Detergent. Disrupts the cell membranes & denatures proteins

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EDTA (Mg2+)

Inhibits DNA-degrading enzymes such as DNase

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<p>NaCl( positively charged salt)</p>

NaCl( positively charged salt)

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

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Tris-HCl

a pH buffer to make it pH (8.0)

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<ol start="2"><li><p>DNA extraction Step 2: Protein Removal</p></li></ol><p></p>

  1. 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

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<p>How is separation achieved?</p>

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

<p>By Centrifuging</p><ul><li><p>spins samples at 1,000 rpm. Forces the precipitate to form a solid pellet (debris pellet)</p></li><li><p>TUBES MUST BE EQUALLY SPACED</p></li><li><p>After that, the supernatant (containing the DNA and some salt/debris) must be transferred to a fresh tube</p></li></ul><p></p>
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<p>Vortex</p>

Vortex

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

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<p>Spin (mini centrifuge)</p>

Spin (mini centrifuge)

spins down PCR tubes for 10-15 seconds

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<ol start="3"><li><p>DNA Extraction Step 3: Alcohol precipitation</p></li></ol><p></p>

  1. 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

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<ol start="4"><li><p>DNA Extraction Step 4: Rehydrate Pellet</p></li></ol><p></p>

  1. 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)