1.  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 Lab 4D (demonstration), Post-Lab Quiz

2. 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

3. Making Solutions for DNA Isolation

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

5.  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

6.  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

7.  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.

8. Pulling DNA out of Solution: DNA Spooling

9.  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

10.  Do calculations in advance—sperm solution concentration is:  Use the C 1 V 1 = C 2 V 2 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

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

12.  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

13.  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

14.  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

15. EtBR Dot Test: A Quick Test for DNA in Samples

16.  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

17.  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