extraction & isolation of plasmid DNA

1. PREPARATION OF PLASMID DNA
Supervision of:
Miss Tehreem Yonus
Presented BY:
• M.Waqas
• Noman Hafeez Khosa
• BS Microbiology (7th Semester)
• Dated: 13-10-2015

2. What do we need DNA for?
•Detect, enumerate, clone genes
•Detect, enumerate species
•Detect/sequence specific DNA regions
•Create new DNA “constructs” (recombinant DNA)

3. cell growth
cell harvest and lysis
DNA purification
DNA purification: overview
DNA concentration

4. Plasmids: vehicles of recombinant DNA
Bacterial cell
genomic DNA plasmids
Non-chromosomal DNA
Replication: independent of the chromosome
Many copies per cell
Easy to isolate
Easy to manipulate

5. E. coli
Chromosome
Plasmids
Plasmids
• DNA molecules separate from chromosomal DNA
• Self-replicating
Electron micrograph of DNA
from a lysed E. coli cell

6. Plasmid purification: alkaline lysis
Alkaline
conditions
denature DNA
Neutralize:
genomic DNA
can’t renature
(plasmids CAN
because they
never fully
separate)

7. DNA purification: silica binding
Binding occurs in presence of high salt
concentration, and is disrupted by elution with
water

8. DNA purification: phenol/chloroform extraction
1:1 phenol : chloroform
or
25:24:1 phenol : chloroform : isoamyl alcohol
Phenol: denatures proteins, precipitates form at
interface between aqueous and organic layer
Chloroform: increases density of organic layer
Isoamyl alcohol: prevents foaming

9. 1. Aqueous volume (at least 200 microliters)
2. Add 2 volumes of phenol:chloroform, mix well
3. Spin in centrifuge, move aqueous phase to a new tube
4. Repeat steps 2 and 3 until there is no precipitate at phase interface
5. (extract aqueous layer with 2 volumes of chloroform)
Phenol extraction

10. cell harvest and lysis
DNA purification
DNA purification: overview
[Used
enzymes
(lysozyme)
reagents for
bacterial
cell lysis]
DNA purification
through “Column
Chromatography”
& “Organic
Extraction”

11. Gel Electrophoresis

12. Definition
• Gel electrophoresis is
a technique used to
separate DNA bands
based on size

13. Agarose
• Neutral
polysaccharide
• Harvested from
Rhodophyceae algal
cell wallshttp://www.steve.gb.com/images/

14. Agarose
• Alternating sugar units
form linear chains
• Cross-linking chains via
hydrogen bonds forms
porous matrix
• Heat breaks matrix apart
• Cooling forms a matrix
with an “average” pore
size
http://www.bioscience-beads.com/imag
Not
necessary
to
describe

15. Gel Electrophoresis
Prepare agarose gel
Melt, cool and add Ethidium Bromide. Mix thoroughly.
Add running buffer, load samples and marker
Run gel at constant voltage until band separation occurs
Pour into casting tray with comb and allow to solidify
View DNA on UV light box and document results