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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
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
slide7
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
slide10
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
slide14
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
slide17
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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