Team:HKU-Hong Kong/Lab Diaries

<LI>Transformation of reporter DNA (pEGFP-loxp-km-loxp) into DH10B (non-virulent strain E. coli) with antibiotic resistance (Chloramphenicol – Cm). To test the efficiency of transformation at different concentration, 3ug/uL, 30ug/uL and 300ug/uL bacterial cell culture were used. 100uL of each sample was used to do spread plates and incubated at 37C overnight. It was found that 3ug/uL of DNA was already enough to have high transformation efficiency of our DH10B competent cells. However, the colonies were very small. This might be because the bacteria grow very slowly. 5 colonies were inoculated from the plate which had been spread with 3ug/uL reporter DNA transformed DH10B separately into 3mL LB broth with 3uL Amp and incubated at 37C for several hours (from ~09:30 to ~14:30) to see if it could really grow in growth medium. Results were failed because DH10B was Cm resistant but not Amp resistant. 5 colonies were inoculated again from the plate which had been spread with 3ug/uL reporter DNA transformed DH10B separately. But this time into 3mL LB broth with 3uL Cm and incubated at 37C overnight. Results were failed again, no turbidity in all the tubes. Perhaps it was because the bacteria grow too slowly. So we put it on the rotary for longer time. There was still no results, no turbidity. It might be because the colonies are so small that when we picked the colonies for inoculation, the bacterial cells could not be picked up by the tips and still stuck to the agar plate; or, the bacterial cells get stuck to the tips without really getting into the LB broth. Inoculation was carried out again as well as plasmid extraction (using mini-prep).</LI>