mohammad tabish ahmed - eTheses Repository - University of ...

Chapter 2
then resuspended in 200µl GTE by pipetting or vortexing. 400µl 1%SDS/0.2M NaOH was
added to lyse the cells (tube inverted several times). 300µl of 3M Na/5m acetate was added to
the solution which was then inverted several times until a white precipitate was formed. The
tube was then incubated for 5 minutes at room temperature and then spun at 13000rpm for a
further 5 minutes. The resulting supernatant, which contains the DNA plasmid, was poured
into a clean labelled Eppendorf and 600µl isopropanol was added (and mixed very
thoroughly). This tube was then cooled at -20°C for 30 minutes before being spun down at
13000rpm for 10 minutes to form a pellet of nucleic acid at the bottom of the Eppendorf. The
pellet was then washed twice with 500µl of 70% ethanol and then again with 500µl of 100%
ethanol (ensuring the pellet was not disturbed). The pellet was then dried at 37°C for 5-10
minutes and resuspended in 50µl TE.
Kit method
Plasmid DNA was purified from E.coli cultures using the Qiagen Qiaprep spin miniprep kit
as per the manufacturers recommended guidelines. 5ml of overnight culture was spun down
at 4000rpm and 4°C for 15 minutes. The pellet was resuspended in 250µl of P1 buffer
solution and transferred to a micro centrifuge tube. The cells were then subjected to alkaline
lysis for 5 minutes by adding 250µl of P2 solution, which degrades bacterial cell walls and
membranes. After this, the reaction was neutralised by the addition of 350µl of N3 buffer.
The cell debris was then spun down by centrifugation at 13000 rpm for 5 minutes. The
supernatants were then added to a Qiaprep spin column and spun at 13000 rpm for 1 minute.
This results in the plasmid DNA binding to the column. The plasmid was then washed in
ethanol and eluted in 50µl of MilliQ water.
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