Team:Groningen/Notebook/Wetwork 7August2012


PCR of Fnr promoter and lacI promoter using Phusion.
PCR of amilCP (blue pigment) and amilGFP (yellow pigment) using Phusion.
PCR of lycopene and violacein using Phusion. Elongation time for this PCR took longer than the other PCR (~2 minutes) due to the size of the pigment coding region (~7 Kb)

- PCR of Fnr and LacI promoter worked, eventhough the fragments did not appear very bright and sharp on the agarose gel.
- PCR of amilCP and amilGFP worked perfectly. ~700 bp DNA fragments were shown on the agarose gel.
- PCR of lycopene and violacein showed unspesific binding (multiple DNA fragments shown on the agarose gel). There was probably a problem with the template that was used for lycopene PCR and higher annealing temperature is probably needed for violacein PCR.

However, PCR product(s) gained from these reactions (Fnr and LacI promoter, amilCP and amilGFP, lycopene from PCR on Saturday) were purified and used for further cloning steps.

Digestion of the promoters, pigments, and backbone pSB1C3 was performed. However unfortunate event happened so ligation and transformation steps could not be performed today.

PCR was performed by using alsT forward primer and GFP reverse primer to check the E.coli that was suspected to contain pSB1C3+alsT promoter+GFP.

- Colony 1 of alsT150 showed positive result (1 Kb)
- Colony 1 and 2 of alsT300 showed positive result (~1.1 Kb). Colony 3 and 4 also showed positive result, but they showed bigger DNA fragments (~1.3 Kb) than colony 1 and 2.

E. coli colonies containing alsT-GFP are grown overnight in LB to be checked tomorrow for its GFP expression

Re-do the cloning work that was performed on Monday.

GC-MS: liquid injection measurements using tolueen as organic solvent. After filtering the tolene an emuslion was seen. injection into the GC-MS did not yield any data.

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