Transformation and inoculation

What is Transformation?

Tranformation is the process of inserting DNA into competent bacteria.

The pBAD and Lysis DNA parts have been ligated and are ready to be inserted back into competent E.coli cells.

The Process:


 * 1) A solution of DH 5-alpha E.Coli cells were added into the plasmid solution.
 * 2) The bacteria 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. The heat shock causes tiny pores in the walls of the bacteria to expand, allowing the plasmids to go through.
 * 3) The Bacteria were then mixed into 330 micro ml LB (Luria Bertani) # The bacteria were then left to cultivate for approximately an hour @ 37 degrees Celsius in the shaker.
 * 4) Centrifuged at room temperature for 3 minutes @ 8000 rpm.
 * 5) Discard 900 micro ml of the supernatant and dissolve pellets in remaining 100 micro ml.
 * 6) These were then spread on LB Agar plates containing 100 micro gram per ml Ampicillin.
 * 7) The plates were incubated at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics, especially ampicillin, start to break down and un-transformed cells will begin to grow.

Once the colones start forming, they must be inoculated.

What is inoculation?

Inoculation is simply putting bacteria in a nutritious substance so that it can cultivate.

We had four batches of the prepared plasmids. One of them had no colonies, while the other three had 18 colonies in total. Each colony was inoculated seperately. The inoculation is done by carefully picking up the colony with the tip of a pippet and dropping the tip into Centrifuge tubes with 5ml of LB with Ampicilin. The tubes are then centrifuged for 12 to 14 hours.