"

From 2010.igem.org

Contents 1 Andreas 1.1 Preparation of competent BL21(DE3) cells 1.2 Preparation of LB agar plates with antibiotics 1.3 Transformations 1.3.1 Top10 1.3.2 BL21(DE3) 1.4 Isolation of requested parts

Andreas

Preparation of competent BL21(DE3) cells

Competent cells were prepared as previously described.

Modifications

• Growth temperature was changed to 37°C
• Final OD(600) was 0.57

Preparation of LB agar plates with antibiotics

• 50 ug/ml Km (20 plates)
• 50 ug/ml Amp & 25 ug/ml Cm (10 plates)
• 50 ug/ml Amp & 25 ug/ml Km (10 plates)

Plates were tested by plating with competent, non-transformed Top10 cells

Transformations

Several different transformations were performed. Top10 cells were transformed with pSB1x3 BioBrick vectors and grown on agar plates with appropriate antibiotic(s). BL21(DE3) cells were transformed with SOD- and yCCS-carrying pEX vectors as well as with the pMA vector to test cell competence and grown on Amp plates:

Top10

• pSB1A3.BBa_J04450
• pSB1C3.BBa_J04450
• pSB1K3.BBa_J04450
• pSB1AC3.BBa_J04450
• pSB1AK3.BBa_J04450

BL21(DE3)

• pEX.SOD
• pEX.yCCS
• pMA.BBa_K157011

Isolation of requested parts

Our three requested parts BBa_J18930 (mCerulean; CFP), BBa_J18931 (mCitrine; YFP) and BBa_J18932 (mCherry; RFP) arrived as agar stabs. These were plated onto Amp plates according to [http://partsregistry.org/Help:Requesting_Parts_2009#Using_agar_stabs Registry instructions].