PLASMID EXTRACTION

Method
Many different methods are available for isolation of plasmid DNA. In this practical you will
be using a commercially available kit, which utilizes an affinity column to purify the plasmid
DNA. Bacteria (E. coli in this case) containing plasmid is grown from a single colony in a
suitable medium such as Luria Broth (LB). An aliquot of the culture is used for preparing
the plasmid DNA.

Centrifugation
Bacterial cult.Centrifugation results in pelleting of bacterial cells

Solution I

(Tris pH 8.0
EDTA- Ethylene diamine tetra acetic acid and
Glucose)
RNase
Lysozyme- optional

· Glucose gives osmotic shock that leads to the rupture
of cell wall and membrane
· EDTA, inhibits nucleases
· Rnase: Dergrades RNA
· Lysozyme is used for rupturing cell wall of bacteria.


Solution II
Sodium hydroxide
SDS- Sodium dodecyl sulphate
(Alkaline pH `12.0

Lyses the cell completely
· Alkaline pH denatures chromosomal DNA but not the
covalently closed circular plasmid DN

Solution III
Sodium or potassium acetate
(Acidic pH 5.4)

· Neutralizes the alkaline pH
· Precipitates protein and forms SDS-protein complex
· Chromosomal DNA renatures and aggregates with
protein

Centrifugation

· Pellets the Protein –DNA aggregates
· Plasmids will be present in the supernatant
(These plasmid can be precipitated with ethanol- This
used to be the conventional method)

Spin column
Silica membrane

· Silica membrane binds to plasmid DNA at high salt
buffer condition

Elution

· Under low salt condition (Distilled water), plasmids
can be eluted from the silica membrane

Protocol
Each group is provided with 10ml of an E.coli (containing plasmid) culture that has been grown
overnight at 37oC.
1. Transfer 1.5ml of bacterial culture to a micro centrifuge tube.
2. Pellet cells by centrifuging at 12,000 rpm for 2 minutes
3. CAREFULLY remove the supernatant.
4. Add another 1.5ml of culture to the same tube and centrifuge and repeat step 2 &3
5. Add 250 ml of Solution I /RNAase.
6. Resuspend the pellet by vortexing briefly or by pipetting up and down.
7. Add 250 ml of Solution II
8. Mix GENTLY by inverting and rotating the tube several times. DO NOT vortex!!!
9. Leave at room temperature for 5 minutes but NOT MORE than 5 minutes.
10. Add 350 ml of Solution III
11. Mix by inverting the tube 6-8 times
12. Centrifuge at 12,000 rpm for 12 min
13. While your tubes are spinning place a Spin column in a 2-ml collection tube.
14. After centrifugation, CAREFULLY transfer the supernatant to the column. DO NOT disturb the
pellet.
15. Centrifuge the column and collection tube at 12,000 rpm for 1 min
16. Discard the flow through collected in the collection tube.
17. Replace the column in the collection tube
18. Add 250 ml of HB Buffer into the column and centrifuge for 1 minute at 12,000 rpm and
discard the flow through.
19. Add 700 ml of wash buffer into the column and centrifuge for 1 minute at 12,000 rpm and
discard the flow through.
20. Centrifuge again to remove residual buffer for 90 seconds
21. Place the column in a sterile 1.5ml tube.
22. Add 50ml of deionised water or TE buffer into the column
23. Centrifuge at 12,000 rpm for 1 min.
24. Discard the column. Plasmid DNA is in the deionised water or TE buffer will be collected in the
1.5ml tube.
25. Place the tube containing plasmid DNA on ice for further use or store at –20oC