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?

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)

Plasmids

Usually occur naturally in bacteria

Circular, ds1-400 kb1- 100/1000 copies per cellSingle OR

Stringent : Replicates only when the host chromosome replicates Relaxed: Replicates independently

Conjugation, a mechanism of horizontal gene transfer.

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

Vectors used in this experimentPKS

PLASMID DNA ISOLATION FROM E.Coli by Alkaline Lysis Method

Lipopolysaccharide

Peptidoglycan

Plasma Membrane

PLASMID

Chromosomal DNA

Proteins

Proteins

Proteins

Proteins

Proteins

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

Table 1. Techniques used for the physical disruption of cells.

Lysis Method Description Apparatus

MechanicalWaring BlenderPolytron

Rotating blades grind and disperse cells and tissues

Liquid Homogenization

Dounce HomogenizerPotter-Elvehjem HomogenizerFrench Press

Cell or tissue suspensions are sheared by forcing them through a narrow space

Sonication SonicatorHigh frequency sound waves shear cells

Freeze/ThawFreezer or dry ice/ethanol

Repeated cycles of freezing and thawing disrupt cells through ice crystal formation

Sol.1- Resuspension Buffer

50mM Glucose Viscosity – to decrease DNA damage !

25 mM TrisHCl

pH 8.0Buffers cells at pH 8 & Maintains osmotic pressure

10 mM EDTABinds divalent cations (Mg++ and Ca++ )

Interferes with cell wall integrity & Inhibits DNases - RNases !

1% SDS Dissolves 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

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)

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.)

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 / dH2O

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)= A260 x 50 x Dilution factor

OD260 = 0.2 0,2 x 50 µg/ml = 10 µg/ml

(2 µlDNA + 98 µl H2O) with 0.2 OD260:

10 µg/ml x 50 = 500 µg/ml

Yield (µg) : 500 (µg/ml ) x Total volume (ml)

Purity

Pure dsDNA A260/A280= 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”

-Nicked-Linear

- Relaxed circular- Supercoiled

1- 1/50 diln in 300 μl dH2O measure OD260!

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!