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From 2010.igem.org

Contents 1 Andreas 1.1 Expression of SOD and yCCS from pEX expression vector 2 Hassan 3 Mimmi 3.1 pMA vector 3.1.1 mini prep

Andreas

Received two cell cultures of DH5alpha cells respectively transformed with pEX vectors carrying superoxidase dismutase (SOD) and yeast copper chaperone (yCCS), prepared by Nina and Johan.

Expression of SOD and yCCS from pEX expression vector

• Set an ON starter culture by inoculating 5 ml LB with 100 ug/ml Amp with 5 ul from each received culture.
  • 37°C, 250 rpm ON.

Continued 30/6

Hassan

"It has become dangerously acceptable to conclude that proteins interact directly provided that (i) the interaction is biologically plausible; (ii) the proteins are co-expressed; and (iii) an interaction is demonstrated by glutathione-S-transferase (GST)-pulldowns and/or co-immunoprecipitations (co-IPs)"... "The most obvious caveat with co-IP data is that a positive result does not imply a direct interaction between two proteins – binding could be mediated by other partners."..."In addition, a widespread misconception is that co-IPs from extracts provide in vivo evidence for the existence of an interaction. This is not accurate, particularly when the experiment is carried out using overexpressed proteins in cell lines."...""

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"Protein interactions: is seeing believing? J. P. Mackay et al. 2007"

Mimmi

pMA vector

mini prep

• See protocol E.T.Z.A. miniprep protocol I

Plasmid conc.	ng/µl
clone A	~160
clone B	~130
control	~1