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

Chapter 2
200µl of 0.9M Na 3 C 6 H 5 O 7 was added and the resultant suspension was spun at 13000rpm for
5 minutes. The supernatant was discarded and the pellet was then resuspended in 100µl LB
and incubated at 37°C for 1 hour. Various dilutions, including neat, were then plated out on
selective media plates e.g. Ampicillin plates to test for Ampicillin resistant gene transfer.
Control plates were also used and included, plates with the recipient strain on its own (not
transduced), as well as the bacteriophage P1 lysate on its own. These plates were incubated at
37°C for 1-2 days. The resulting colonies were screened by colony PCR.
2.4.6- Plasmid purification (Mini-prep)
Two methods of miniprep plasmid extraction were used during the project to extract plasmid
DNA from E.coli.
Alkaline lysis method
Reagents required:
- GTE (50 mM glucose, 25 mM TRIS pH8.0, 10 mM EDTA) – autoclaved before use
- 1% SDS/0.2M NaOH (from stock of 10% SDS and 10M NaOH)
- 3M Na/5M acetate
- TE (10 mM TRIS pH8.0, 1 mM EDTA) – autoclaved before use
- Isopropanol
- 70% ethanol (in TE)
- 100% ethanol
1-5mls of overnight culture was centrifuged at 13000rpm for 2 minutes and the supernatant
was poured off. The culture was then spun again for 20-30 seconds and the remaining LB
was removed using a Gilson pipette. This is an important step as the left over LB can carry
components into the prep, which can inhibit restriction enzymes and ligases. The pellet was
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