DNA extraction

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DNA extraction
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1. Chew your cheek cells, swish with 3 ml of
distilled water, expectorate the water and cheek
cells back into the tube
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Add lysis buffer 2ml

• WHY?

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What is Lysis Buffer? 50 mM Tris-HCI, pH 8.0
1 SDSTris buffer to maintain the pH of the
solution at a level where DNA is stable1 SDS
to break open the cell and nuclear membranes,
allowing the DNA to be released into the
solution(SDS also functions to denature and
unfold proteins, making them more susceptible to
protease cleavage).
Why add lysis buffer?
Na
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2. Add protease 5 drops

• WHY?

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3. Why Add Protease?

• Protease is added to destroy nuclear proteins
  that bind DNA and cytoplasmic enzymes that
  breakdown and destroy DNA.
• Protease treatment increases the amount of
  intact DNA that is extracted.

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Included in the protease was 5M NaCl.

• WHY?

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Adding Salt(5 M NaCI)

• Na ions of NaCI bind to the phosphate groups
  of DNA molecules, neutralizing the electric
  charge of the DNA molecules.
• The addition of NaCI allows the DNA molecules
  to come together instead of repelling each other,
  thus making it easier for DNA to precipitate out
  of solution when alcohol is added.

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(5 M NaCI)
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Incubate at 50o C for 10 minutes

• WHY?

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Why incubate?

• Think about it!!!

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The DNA is then put in cold alcohol for 5 minutes

• WHY?

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5. Adding Ice Cold Alcohol?

• DNA cannot dissolve in alcohol.
• The addition of cold alcohol makes the DNA clump
  together and precipitate out of solution.
• Precipitated DNA molecules appear as long pieces
  of fluffy, stringy, web-like strands.
• Microscopic oxygen bubbles aggregate or fuse
  together, simultaneously with the DNA
  precipitation.
• The larger, visible air bubbles act to lift the
  DNA out of solution, from the aqueous into the
  organic phase.