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

Chapter 2
2.4.7- Plasmid maxi prep
This method of plasmid prep was used on large volumes of culture to extract a large amount
of plasmid. Plasmid maxi prep was done using the GenElute plasmid maxiprep kit by Sigma-
Aldrich as per the manufacturer’s guidelines. A total of 50ml of overnight culture was
centrifuged at 5000rpm for 10 minutes obtain the cell pellet. The pellet was resuspended in
6ml resuspension solution provided with the kit and transferred into a Falcon tube. To this
6ml of lysis solution was added, mixed thoroughly, and allowed to clear for 5 minutes. Once
the solution had cleared, 8ml of neutralization solution was added and the tube was inverted
4-6 times. This was then centrifuged at 15000g for 15 minutes. The resultant supernatant was
then transferred to the maxi spin column in a 50ml collection tube and spun in a swing bucket
rotor at 4000g for 2 minutes. The flow through was discarded, 15ml of washing solution was
added to the column and the tubes were spun again at 4000g for 5 minutes. The column was
then transferred to a clean collection tube, 5ml of elution buffer was added, and the tube was
spun at 4000 g for a further 5 minutes. The flow through obtained via this method contained
the plasmids from the overnight cultures and was stored at -20° C.
2.4.8- Restriction digests of plasmids
For the restriction digest of a plasmid, approximately 300ng of plasmid DNA was used. For
all digests the restriction enzymes and buffers used were either from New England Biolabs or
Fermentas (20,000 units/ml) and approximately 2000 units of restriction enzyme was used in
each case for complete digestion. The following contents were used to make a total volume of
20µl:
- Milli Q water 5-15 µl
- NEBuffer (depending on the restriction enzyme) 2 µl
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