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

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

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

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

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

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

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

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

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

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

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.

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