Team:Paris Bettencourt/Notebook/Drug Screening/Thursday 4th July.html

Drug Screening

ASDF

Thursday 4^th July

Heat Shock Transformation

Preparation of Agar Plates
LB Agar was melted; 100ml was used to make 8 ampicillin plates,
100ml was used to make 8 spectinomycin plates and the remaining was used to make 20 chloramphenicol plates.
Antibiotic concentrations are: Ampicillin 50ug/ml, Spectinomycin 50ug/ml, Chloramphenicol 34ug/ml.

Heat Shock Transformation
sD001: BL21 (DE3) with deletions, was transformed with the following plasmids and plasmid combinations:

• sD001+pD004 ( FNR, Spectinomycin)
• sD001+pD005 ( SIR, Fdx, Chloramphenicol)
• sD001+pD006 (Sir, Chloramphenicol)

Transformation was done according to protocol 1 with the following specifications:
BL21 (DE3) chemically competent cells were thawed on Ice. 1ul of plasmid DNA was added to a tube of BL21 (DE3) cells.
Cells were incubated on ice for 30 minutes. Cells were transfered to heating block at 42C for 45s.
Cells were returned to ice for 2 minutes. 1ml of LB Broth was added to the cells and they were incubated at 37C for 1h.
Cells were then plated on agar with appropriate antibiotics (specified previously). These transformations worked; single colonies were lifted from plates, entered to catalog and stocked in glycerol stock (protocol 3).
Catalog names:

• sD006= sD001+pD004 ( FNR, Spectinomycin)
• sD007= sD001+pD005 ( SIR, Fdx, Chloramphenicol)
• sD008= sD001+pD006 (Sir, Chloramphenicol)