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From 2012.igem.org

Emeraldo/Tom

Ligation of alsT 300, Fnr and s-box promotor to GFP and lycopene in Psac with a new protocol. Psac was digested with EcorI. Fnr, alsT 300 and s-box were digested with EcorI and SpeI. Lycopene and GFP were digested with PstI and XbaI. Reaction was incubated for 2 hours at 37 degrees celcius.

The different restricted DNA fragments were analysed on an agarose gel and removed from it. The obtained gel fragment was purified. For the ligation an end volume of 10 ul was used. 8.5 ul was used for the backbone and the template. Approximately ratio of backbone and template was 2:3:3. Ligation was incubated at 22 degrees celcius for two hours. Obtained DNA was transformed into E. coli.


Tom/Renske

PCR amplification of promoters+GFPopt:
1) AlsT 300_GFP: FW Alst300 + RV GFP
2) Alst 500_GFP: FW Alst500 RV GFP
3) combine FNR+GFP: FW FNR, RV GFP
4) AlsT250: FWAlst250_RV GFP
5) AlsT150: FW Alst150, RV GFP


Yonathan
Another round of incorrect Lycopene PCR products. Concluded that either the primers are not correct or the brick is not correct. Decided to sack this pigment as an idea for work flow reasons. Back to notebook