Han Xiao PhD thesis - Research@StAndrews:FullText - University of ...

2.3.3 Transformation of competent cells
0.1μg plasmid was added directly to 50μl of thawed, chemically competent cells
(Invitrogen). After incubation on ice for 30min, cells were heat-shocked at 42°C in water
bath for 30sec before being immediately immerged in ice for a further 2min. Cells were
resuspended in 1ml LB broth and incubated at 37°C for 1h. The cell suspension was
plated out onto LB-agar plates supplemented with ampicillin (90 mm-diameter Petri
dishes; Scientific Laboratory Supplies Ltd., U.K.). Plates were inverted and incubated at
37°C overnight.
2.3.4 Preparation of plasmid DNA
For small-scale preparations, bacterial cell cultures of 10 ml (in LB broth containing
100μg/ml ampicillin) were grown overnight in a 37°C shaking incubator. DNA was
extracted from cells and purified using the QIAGEN DNA mini-prep kit according to the
manufacturer’s instructions (QIAGEN). To produce larger-scale DNA preparations, 100
– 500 ml of bacterial culture was grown overnight in a 37°C shaking incubator. DNA was
extracted from cells using the QIAfilter Plasmid Maxi Kit according to the
manufacturer’s instructions (QIAGEN).
2.3.5 Colony PCR screening
Colony PCR screening was used to quickly test for the presence of a target DNA
fragment in E.coli without the need for growth and mini-prep of transformed bacterial
colonies. Basically, a master mix of PCR reaction with primers for gene of interest was
prepared and dispensed into PCR tubes. Bacterial colonies were picked up individually
and resuspended into the PCR reaction tube, and PCR amplification was performed at
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