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Lab 4A

Lab 4A. Making Solutions for DNA Isolation. Part 1: Preparation of 5 M of NaCl. Show calculations involved in lab notebook Draw diagram of how solution is prepared in notebook Store finished mixture at 4 degrees Celsius until ready for use. Part 2: Preparation of TE Buffer.

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Lab 4A

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  1. Lab 4A Making Solutions for DNA Isolation

  2. Part 1: Preparation of 5 M of NaCl Show calculations involved in lab notebook Draw diagram of how solution is prepared in notebook Store finished mixture at 4 degrees Celsius until ready for use

  3. Part 2: Preparation of TE Buffer • Show calculations for TRIS and EDTA mass in lab notebook • Draw a diagram of how the TE buffer solution is prepared in notebook • For pH adjustment: • Slowly add HCl to lower pH • Slowly add NaOH to raise pH

  4. Overview • Make 10 mL of 5 M NaCl solution • Make 100 mL of TE buffer • Keep buffer at 4 degrees Celsius until ready for use • 1 mM TRIS • Maintains pH of the DNA sample • 1 mM EDTA • Denatures Dnases and keeps the DNA sample

  5. Tips Students who finish early should make extra TE buffer for later use Analytical balances may be used for TRIS and EDTA measurements 5 M naCL is very concentrated; it may take a long time for the sodium chloride to fully dissolve.

  6. Lab 4B Pulling DNA out of Solution: DNA Spooling

  7. Overview • DNA can be pulled out of aqueous solutions due to certain molecular characteristics: • Long double helix shape • Charged phosphate groups • Repelled by nonpolar solutions, such as ethanol

  8. Tips • Ethanol is flammable; keep away from open flame. • Use the C1V1 = C2V2 equation to determine correct solutions • Record calculations and data in lab notebook • When adding ethanol to the DNA/salt mixture, it is very important to have two distinct layers. • Trickle ethanol down the side of the beaker

  9. Lab 4C Testing for the Presence of DNA, RNA, and Protein in DNA Extracts

  10. DPA Solution • Diphenylamine (DPA) solution is used as an indictor for DNA • Turns blue when DNA is present • Turns green when RNA is present • Indicates that the DNA sample is contaminated • Turns brown or blue-green if both DNA and RNA are present

  11. Biuret Indicator • Tests for the presence of protein • Positive test results range from medium blue to violet • Indicates that the DNA sample has yet to be completely purified

  12. Tips DPA produces toxic fumes and should only be used in a chemical fume hood with a fan running Poor spooling technique may contaminate DNA sample with alcohol, which will yield inaccurate results

  13. Lab 4D EtBR Dot Test: A Quick Test for DNA in Samples

  14. Overview • EtBr (ethidium bromide) indicates the presence of DNA by intercalating between nitrogenous base pairs • Causes the bond angles to change, producing different light-absorbing patterns • UV light that shines on an EtBr and DNA mixture will glow pink-organe • Intensity of glow indicates the presence and amount of DNA

  15. Tips • Ethanol contamination of samples can result in poor EtBR dot test results • EtBr is known to change the shape of DNA • Therefore, it is a suspected mutagen and carcinogen • Wear goggles and gloves when in near proximity to EtBr

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