Preparation of chemically competent DH5αTM E.coli cells (Thermo Fischer Scientific)

Prepared LB, SOC media and LB plates with different antibiotic markers. Liquid Media Left under foaming hood at Room temperature after autoclaving, plates in fridge or cooling room at 4°CBacterial cells were cultured in 2 ml LB over-night at 37°C shaking at 180 RPM.1 ml of the over-night culture were then diluted up to 200 ml of LB medium, theculture was subsequently grown up to an O.D600=0.6 (about 2.5-3 hours). Subsequently the cultured bacteria were pelleted and re-suspended in 15 ml TSS buffer (1XLB, 5% DMSO, 10 mM MgCl2, 10 mM MgSO4, 10 mM PEG 4000). The TBS (TSS) bacterial suspension were left on oce to cool down for 10 minutes. The suspension was divided into individual 100 μl aliquots that were kept ondry ice and subsequently preserved at -80°C.

2 Batches were prepared in parallel.

Batches were both tested next day by transforming 3 aliquots each with BBa_J04450. Alliquots were thawed on ice and after gently mixing by pipetting up and down split into 2*50 μl (for negative control). 1 or 10 pg DNA were added and mixed by gently flicking tubes. Protocol for Transformation of bacterial cells with non-ampicillin-antibiotic marker was followed.

Red colonies were counted to get following average efficiencies:

Batch 1:   \(7,1*10^6 \frac{cfu}{μg}\)

Batch 2:   \(1,2 *10^7 \frac{cfu}{μg}\)