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Rensselaer/30 July 2008
From 2008.igem.org
Promoter Group - 7.30.08
Transformation of Fe Promoter (Plate 1007 Well 7C) Into TOP10 E. coli
Set up petri dishes with bleach, dH2O, ethanol for wash of punch tool.
Aliquot 10 uL H20 to PCR tube. Incubate 10 uL H2O @ 50C for 5 minutes.
Punch out two DNA disks into 10 uL H2O making sure to wash punch tool in between.
Incubate PCR tube w/ H20 and DNA @ 42C for 20 minutes.
During incubation, thaw TOP10 supercompetent cells on ice.
Transfer all liquid to TOP10 cells. Incubate on ice 30 minutes.
Heat shock cells @ 42C 45 s. Incubate on ice for 2 minutes.
Transfer sample (cells + DNA) to 900 uL 2XYT. Incubate 1h @ 37C w/ shaking.
Plate 100 uL undiluted and 100 uL 50% concentrated on LB + 100 ug/ml amp plate.
Incubate @ 37C ON.
DNA Miniprep
Result:
Pelleted 5 ml of cells with Ptet promoter and mRFP reporter (plasmid part BBa_J61002).
Plasmid was renamed to pPDJ, stored in 100 ul of H2O.
Carotenoid Group 7.30.08
Transformed BL21 cells were examined. pUC-MN plasmid produced slightly yellow cells. pUC-EBI plasmid produced pale pink colonies with some brighter pink colonies. pUC-crtY cells showed normal coloration.
The two different observed colony types in pUC-EBI were streaked out on a plate for comparison. Colonies from all three transformations were transferred to liquid LB and shaken at 37C overnight to prepare for plasmid minipreps and freezerstocks.
