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Double Terminator

The DNA was extracted from lanes 3&4 of the gel and extracted using the Zyppy Gel Extraction kit. The extracted DNA was then PCRed using 34 cycles, and then 10μL was frozen at -20°C. 8μL of the PCR product was cut with both EcoR1 and Pst1, as was 5μL of the death plasmid, PSB4C5, using the fast digest protocol. The gene was then ligated into the plasmid backbone, using the fast ligation kit, from Finnzymes.

500μL of normal TOP10 cells were made competent, and then transformed with the 4μL of the new plasmid, and grown on chloramphenicol overnight. 1μL of Puc19 was used as a control.

K+ assay for E.coli

Solutions of potassium chlotide were made up from 0mM to 500mM. 100μL of cells, 100μL of 10x KCl solution and 800μL of SDW were mixed and the OD600 measured every 30 mins, after agitation to ensure homogenaity. After 4 hours 800μL of the cell solution were spun down and resuspended in 1mL SDW, lysed using the freeze-thaw method, and the K+ concentration measured using the Flame Photometer.