Bacterial Transformation : The Process
4 micro test-tubes were taken and marked lysis, p-tet, positive and negative. Fist we extracted the dry DNA from the DNA Distribution Kit by mixing each part with 15 micro ml of water using a micro-pipette. 2-3 micro ml of this plasmid solution was then added to 100 micro ml of DH 5-alpha E.Coli cells. The positive and negative marked tubes were also filled with control plasmids.
The tubes were incubated in ice for 40 minutes, then given a heat shock for 30 seconds @ 42 degrees Celsius and then put in ice for 2-5 minutes.
After this, they were mixed with 330 micro ml LB (Luria Bertani) and left to grow for approximately an hour @ 37 degrees Celsius in the shaker after which they were centrifuged at room temperature for 3 minutes @ 8000 rpm.
The next step was to discard 900 micro ml of the super-natured solution and dissolve pellets in remaining 100 micro ml. These were then spread on LB Agar plates containing 100 micro gram per ml Ampicillin.
Now the plates were incubated at 37 degrees Celsius overnight.