Plasmid

Purification
Objective:
To purify plasmid DNA and to verify
the presence of an inserted gene in
the plasmid.
Procedure:
1. From a culture inoculated in an LB
medium by bacterial colony,take
1.5ml of bacterial culture and
centrifuge for 5 minutes 5000rpm (to
separate bacteria in pellet from
supernatant).
2. The pellet is resuspended in
solution 1 which is GTE by which one
of its components is EDTA used for
chelation of Mg2+ to prevent

inactivation of DNase and Ca2+ to

reduce stability of membrane.
3. Add lysozyme to degrade the
peptidoglycan of bacteria(cell wall).
4. Cool for 5 minutes at room
temperature.
5. Add solution 2 that contain
NaOH(to increase the pH rendering it
basic medium where the double
stranded DNA will be uncoiled into
single stranded) and SDS(to degrade
bacterial membrane).
6. Incubate in ice in order to block all
other enzymatic reactions with the
only variable being the pH.
7. Neutralize the solution by adding
potassium acetate for the
renaturation of plasmid (plasmid
supercoiled so it can be
renatured).Also the potassium acetate
will precipitate all proteins except for
plasmids.

8. Take supernatant that consist the

plasmid.
9. Add isopropanol for precipitating
nucleic acid.
10. Centrifuge for 20 minutes to
precipitate all nucleic acid.
11. Keep the pellet and decant the
supernatant .
12. Add ethanol (to get rid of traces of
isopropanol)
13. Centrifuge then decant
supernatant.
14. Remove traces of ethanol by
putting the tube containing pellet in
oven.
15. Add TE for chelation ;to ensure that
all DNase is removed.