1. Quiz 1.Bacteria of which phase are used for most experiments? Why? Which phase today? 2. What are the three properties that a plasmid has to have to be useful for cloning purposes? And guess the fourth property to be used for protein expression purposes?

2. If you repeat the “bacterial growth curve” experiment, which step would you do different than you have done last week? (To perform one of your data collection step correctly and easier)

3. Plasmids Usually occur naturally in bacteria Circular, ds 1-400 kb 1- 100/1000 copies per cell Single OR Stringent : Replicates only when the host chromosome replicates Relaxed: Replicates independently Conjugation, a mechanism of horizontal gene transfer.

4. Vectors Plasmids used in genetic engineering (as vectors) Antibiotic resistance gene MCS (multiple cloning site) OR (origin of replication) Commercially available for cloning purposes Commonly used to multiply (make many copies of) or express particular genes

5. Vectors used in this experiment PKS

6. PLASMID DNA ISOLATION FROM E.Coli by Alkaline Lysis Method Lipopolysaccharide Peptidoglycan Plasma Membrane PLASMID Chromosomal DNA Proteins

7. Plasmid DNA Isolation (Alkaline Lysis Method) o/n culture of cells containing plasmids (liquid medium) Cell extract preparation by SDS Lysis Deproteinization by Phenol Extraction Removal of salts and concentrating the DNA Miniprep (ng)---2 ml Midiprep (μg)---10ml Maxiprep (mg)---50ml

8. Table 1. Techniques used for the physical disruption of cells. Lysis MethodDescriptionApparatus Mechanical Waring Blender Polytron Rotating blades grind and disperse cells and tissues Liquid Homogenization Dounce Homogenizer Potter-Elvehjem Homogenizer French Press Cell or tissue suspensions are sheared by forcing them through a narrow space SonicationSonicator High frequency sound waves shear cells Freeze/Thaw Freezer or dry ice/ethanol Repeated cycles of freezing and thawing disrupt cells through ice crystal formation

9. Sol.1- Resuspension Buffer 50mM GlucoseViscosity – to decrease DNA damage ! 25 mM TrisHCl pH 8.0 Buffers cells at pH 8 & Maintains osmotic pressure 10 mM EDTA Binds divalent cations (Mg ++ and Ca ++ ) Interferes with cell wall integrity & Inhibits DNases - RNases ! 1% SDSDissolves the phospholipids Denatures protein components of the cell membrane (- charge)  Lysis cell memb. 0.2 N NaOH* pH 12: Denature protein components of the cell membrane * Denatures chr. DNA into single strand and the supercoiled plasmid (but still circular) Sol. 2- Lysis Buffer

10. Sol. 3- Neutralizing Buffer 3 M Na-Acetate pH 5.0 1- Insoluble precipitate of SDS/lipid/protein complex Plasmid in suspension Chromosomal DNA + SDS-protein Complex 2- Neutralizes the sodium hydroxide !!! Chr. DNA (mesosome attached) in the SDS/lipid/protein precipitate (ss) (Cell debris + chr DNA – mesosome attached) !!! The plasmid DNA renatures & In the supernatant (ds)

11. Phenol-chloroform-isoamylalcohol extraction (25:24:1) Phenol: Dissociate proteins from nucleic acids Chloroform: Protein and lipid denaturation Isoamylalcohol: Prevents foaming Biphasic mixture Organic phase : Proteins Aqueous phase (upper) : Nucleic acids (+ other contaminants such as salts, sugars, etc.)

12. Ethanol Precipitation of DNA & Concentrating DNA is polar, soluble in water & Insoluble in less polar ethanol 1. 100 % ethanol & -70 Centrifugation Precipitate DNA & salts that form ionic bonds with DNA !!! Ethanol interaction with the water  Less water molecules are free to dissolve DNA 2. 70% ethanol 30 % water solubilizes the salts present in the pellet !!! Supernatant is removed DNA is resuspended in TE / dH 2 O

13. Quantification of the Plasmid DNA Agarose gel: Comparing the intensity of the ladder bands (for linear DNA) Spectrophotometry: OD at 260nm 1 OD = 50 µg/ml ds DNA = 37 µg/ml ss DNA = 40 µg/ml ss RNA Q (µg/ml)= A 260 x 50 x Dilution factor

14. OD 260 = 0.2  0,2 x 50 µg/ml = 10 µg/ml (2 µlDNA + 98 µl H2O) with 0.2 OD 260 : 10 µg/ml x 50 = 500 µg/ml Yield (µg) : 500 (µg/ml ) x Total volume (ml)

15. Purity Pure dsDNA A 260 /A 280 = 1.7-1.9 (1.8) OD 260/280 < 1.8  protein contamination (Prt absorbs at 280nm) OD 260/280 > 1.8  RNA or residual organics contamination OD 260/270 ratio should be ~1.2, if no phenol cont. OD 260/280: 2.0  Phenol OD 260/230  2 < 2  presence of organics OD 330 should be “0”

16. -Nicked -Linear - Relaxed circular - Supercoiled

17. 1- 1/50 diln in 300 μl dH2O  measure OD 260 !  1μl using Nanodrop Spectrophotometer! 2- 5 μl plasmid DNA + 1 μl 6X agarose loading dye 4 μl 1 kb ladder (Fermentas SM0313) 1% agarose gel Take the image of agarose gel separated plasmids!