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4A, B, C, D Timeline. Monday, October 1 st DNA & RNA Lecture Tuesday, October 2 nd Pre-Lab Quiz, Lab 4A (if time) Thursday, October 4 th Lab 4B (50 minutes) Monday, October 8 th MAGIC MOUNTAIN! Tuesday, October 9 th Step 12 from Lab B, Lab 4C Thursday, October 11 th

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4A, B, C, D Timeline

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4a b c d timeline

4A, B, C, D Timeline

  • Monday, October 1st

    • DNA & RNA Lecture

  • Tuesday, October 2nd

    • Pre-Lab Quiz, Lab 4A (if time)

  • Thursday, October 4th

    • Lab 4B (50 minutes)

  • Monday, October 8th

    • MAGIC MOUNTAIN!

  • Tuesday, October 9th

    • Step 12 from Lab B, Lab 4C

  • Thursday, October 11th

    • Lab 4D (demonstration), Post-Lab Quiz


4a b c d prep

4A, B, C, D Prep.

4A

Check supplies: sodium chloride, TRIS, EDTA, pH paper, HCl & NaOH

4B

Check supplies: salmon sperm DNA,

Get ice

4C

Check supplies: salmon sperm DNA, gelatin, DPA, sulfuric acid, acetic acid, acetaldehyde, NaOH 10%, Cupric Sulfate 5%

Prepare NaOH 10% and Cupric Sulfate

4D

Dilute ethidium bromide


Lab 4a

Lab 4A

Making Solutions for DNA Isolation


Overview

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


Part 1 preparation of 5 m of nacl

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

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


4a b c d timeline

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 NaClis very concentrated; it may take a long time for the sodium chloride to fully dissolve.


Lab 4b

Lab 4B

Pulling DNA out of Solution: DNA Spooling


Overview1

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


4a b c d timeline

Tips

  • Do calculations in advance—sperm solution concentration is:

  • Use the C1V1 = C2V2 equation to determine correct solutions

    • Record calculations and data in lab notebook

  • Prepare data table in advance

  • Keep samples on ice

  • Ethanol is flammable; keep away from open flame.

  • We will finish step 12 on Tuesday

  • 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


Lab 4c

Lab 4C

Testing for the Presence of DNA, RNA, and Protein in DNA Extracts


Dpa solution

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


Biuret indicator

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


4a b c d timeline

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


Lab 4d

Lab 4D

EtBR Dot Test: A Quick Test for DNA in Samples


Overview2

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


4a b c d timeline

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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