Question

This is what we did in the experiment .First of all,1 µl of plasmid DNA was added into the tube which contains competent cells and the tube was tapped gently to mix DNA and the competent bacteria. After that it was placed on ice for 30 minutes. Then, the tube with the competent bacteria and plasmid were transferred to heating block at 42 °C and the tube was leaved in there exactly 90 seconds. 0.25 ml of LB broth was added to the tube and incubated 20 minutes at 37°C . After that, with using aseptic techniques pipette, the entire contents of the 500 µl X-gal/IPTG vial was put onto the agar plate which is labelled and with using glass spreader, it was dispersed. 200µl of bacteria which has the plasmid was spreaded onto the agar plate with glass beads. Then, the plate was inverted and incubated 37°C overnight and one week later the results were observed and there were some blue colonies on the agar plate.

what do the control plates prove to you?did you see only blue colonies or were there any white ones as well and why?

THURSDAY 1430 Control plates competent OH LB + Amp Emperor Thursday Eelso fropsunul Eolso Astia tua sadu, No Growth Growth
can you explain these different plates?

Answer 1

LB+ Amp plates - Blue colored growth of transformed E. coli

LB+ Amp plates - No growth of competent E. coli

LB only- Growth of competent E. coli

The competent cells of E. coli which were successfully tranformed with plasmid became resistant to ampicilling and thus grew on the plate. On LB agar, competent E. coli cells were able to grow. On LB+Amp agar, only competent (non-transformed) E. coli cells which were susceptible to ampicillin could not grow.

Blue colonies in blue white screening represent that the desired fragment is not ligated completely in competent cells. White colonies represent desired fragment is ligated in competent cells.

If cells are completely transformed, they will look white and will grow on ampicillin. But, if desired fragment is not ligated completely and only fractional transformation has taken place, cells will grow on amp agar but will appear blue.