1. Plasmid Miniprep

2. Broad and Long Term Objective To characterize a single clone from an To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis

3. Research Plan Preparation of Competent Cells/Bacterial Transformation Growth of Transformant and Plasmid MiniPrep DNA Sequencing Sequence Analysis

4. Today’s Laboratory Objectives  To isolate high quality plasmid DNA that can be used as template for DNA sequencing  To quantify and determine the purity of the isolated plasmid DNA  To determine the size of the plasmid DNA and its insert

5. Map of Parent Vector pMAB58

6. Theoretical Basis of the Alkaline Lysis Plasmid Miniprep 1. Lyse Cells 2. Separate nucleic acids from other cellular macromolecules 3.Concentrate nucleic acids 4.Separate RNA from DNA

7. 1. Lyse Cells Alkaline/SDS Cell Lysis SDS: anionic detergent that solubulizes membranes and denatures proteins Sodium hydroxide: pH to 12.0, denaturation of DNA

8. NaOH K-acetate + protein 2. Separation of nucleic acids from other cellular macromolecules

9. 3. Concentration of Nucleic Acids Precipitation 1: Isopropanol + cations (Na + ) Precipitation 2: PEG + cations (Na + ) (increased purity for sequencing)

10. 4. Separation of RNA from DNA RNaseA

11. Theoretical Basis of UV Spectrophotometry  A UV spectophotometer measures the amount of light particular molecules absorb (Proteins at 280 nm; Nucleic acids at 260 nm)  Lambert-Beer law describes the relationship between absorbtivity coefficient and concentration and is given by the following equation: A=εbc Where: b= light path length (cm) c=concentration of substance (ug/ml) (ug/ml) ε=extinction coefficient For DNA the extinction coefficient, ε= _____1_____ coefficient, ε= _____1_____ 50 ug/ml * cm 50 ug/ml * cm

12. Theoretical Basis of UV Spectrophotometry To Quantify your DNA sample*: Abs 260nm x Dilution Factor x 50 ug/ml= concentration of DNA in a sample using a 1 cm pathlength Abs 260nm x Dilution Factor x 50 ug/ml= concentration of DNA in a sample using a 1 cm pathlength To estimate the purity of your sample*: A260/A280= ratio of nucleic acids/protein A260/A280= 1.6-1.8 is optimal for DNA

13. Theoretical basis of restriction endonuclease digestion Type II restriction endonucleases cleave Type II restriction endonucleases cleave DNA at specific, usually palindromic, DNA at specific, usually palindromic, sequences sequences

14. Theoretical Basis of Agarose Gel Electrophoresis  Agarose is a polysaccharide from marine algae that forms a matrix allowing separation of DNA molecules  Because DNA is a (-) charged molecule, when subjected to an electric current it will migrate towards a (+) pole  Separation based upon DNA size and secondary structure

15. Pouring an Agarose Gel 12 3 456 78 9

16. Sizing a Piece of DNA  Size of DNA molecule can be determined by using standards of known size * 1. A standard curve is made by plotting the size (in bp) of the standards (Y-axis) against the distance bp) of the standards (Y-axis) against the distance each fragment has migrated from the well (X- each fragment has migrated from the well (X- axis) axis) 2. Measure the distance the unknown fragment migrated from the well 3. Substituting the distance the unknown migrated into the equation of the line of best fit, and into the equation of the line of best fit, and solving for Y (the size) solving for Y (the size)