Download Rop protein

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, 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.
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 experiment
PKS
PLASMID DNA ISOLATION FROM E.Coli
by Alkaline Lysis Method
Proteins
Proteins
Proteins
Proteins
Proteins
Plasma Membrane
Peptidoglycan
Lipopolysaccharide
PLASMID
Chromosomal DNA
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
Mechanical
Waring Blender
Polytron
Rotating blades grind and disperse
cells and tissues
Liquid Homogenization
Dounce Homogenizer
Cell or tissue suspensions are
Potter-Elvehjem
sheared by forcing them through a
Homogenizer
narrow space
French Press
Sonication
Sonicator
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
Sol.1- Resuspension Buffer
50mM Glucose
Viscosity – 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 !
Sol. 2- Lysis Buffer
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. 3- Neutralizing Buffer
3 M Na-Acetate pH 5.0
1- Insoluble precipitate of
SDS/lipid/protein complex
Plasmid in
suspension
2- Neutralizes the sodium hydroxide
Chromosomal DNA
+
SDS-protein Complex
!!! 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!