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