DNA

1. Long term storage of genetic information Double Helix Made up of nucleotides A, T, G, C Supercoiled to allow for efficient storage DNA

2. Isolation of Macromolecules • Rupturing Cells • Detergents • SDS • Disrupts all membranes • Mechanical shearing • Allows cell membrane to be fractured without disrupting other membranes • Use of enzymes • Phospholipase

3. Isolation of Macromolecules • Isolation by separation of internal compartments • Centrifugation • Pellet- solid portion of dense cell debris • Supernatant- liquid portion that contains less dense cell components • Density Gradient • Can be sucrose or Cesium Chloride • Cell components separated by density in a column • Filtering through cloth

4. Isolation of Macromolecules • Isolation by exclusion • Degradation of other molecules • Proteases- degrade protein • DNAses-degrade DNA • RNAses- degrade RNA • Phospholipases- degrade phospholipids • Separation by polar/non-polar separation • Phenol extraction- removes non-polar molecules

5. Isolation of Macromolecules • Isolation by precipitation • Nucleic Acids- precipitate in high salt solutions • Very effective in the presence of alcohol • Proteins- precipitated by acetone and various other chemicals • Precipitated molecules are collected by centrifugation and resuspended

6. Measuring Concentration of DNA • WEAR GLOVES FOR THE WHOLE LAB! • DO NOT MOUTH PIPETTE! • Each group will have 4 tubes + 1 unknown • Using the 4 tubes prepare the standard indicated on the table in lab handout. Prepare unknowns- add 4mL of diphenylamine to unknown sample • Label tubes well- on top of tube • Only 1 group needs to make Blank • Boil Samples for 15 minutes

7. Isolation of DNA • While DNA is boiling move on to isolation of DNA • Add 10mL of detergent to cup of strawberries, and crush with another conical tube • Put a square of cheese cloth on a 50mL falcon tube and filter liquid • Add 2mL of 2M NaCl • Calculate 70% of the volume of the solution • Add that amount of Isopropanol • GENTLY invert • Observe the DNA- looks like spit • Write observations in Lab report

8. Measuring Concentration of DNA • After boiling tubes should be blue • Each group will get 3 cuvettes • 1 for the unknown • 1 for the standards • One for the blank • Each cuvette holds 1 mL • Measure concentrations of standards from lowest to highest concentration • Repeat each measurement 3X • Write results in projected excel spreadsheet

9. Measuring Concentration of DNA • Use class data for standards • Use to calculate S.D., S.E., and Mean • Identify unknown concentration using standard curve

10. Lab Report • Intro: • Brief background and hypotheses • Materials and Methods • Summary of protocols followed, include volumes, reagents used and which unknown you had • Results: • Results of DNA precipitation • Table of class data with calculated mean, SD, & SE • Standard curve generated from class averages with error bars

11. Lab Report • Discussion • Analyze your results for each experiment • What was your estimated unknown concentration • Troubleshoot if necessary • Conclusion • Summary of experiment • Hypotheses supported? Why or why not.

12. Due Next Week • Lab report on Lab #2 due Thursday • Start it tonight so that you can ask questions in class tomorrow if need be