Bacterial Transformation : The Process

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1.The eppendorf tubes being labeled
2.The dry DNA being extracted
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4.The four eppendorf tubes being cooled
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  1. Four eppendorf tubes were taken and marked lysis, p-tet, +ve and -ve. The positive control had the gene that promoted Ampicillin resistance and the negative control did not. If any colonies started forming in the negative control batch, that would mean the entire batch is contaminated.
  2. The dry DNA from the DNA Distribution Kit was extracted by mixing each part with 15 micro ml of water using a micro-pipette.
  3. 2-3 micro ml of this plasmid solution was then added to 100 micro ml of DH 5-alpha E.Coli cells.
  4. 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.
  5. The Bacteria were then mixed into 330 micro ml LB (Luria Bertani) which is a source of required nutrients.
  6. The bacteria were then left to cultivate for approximately an hour @ 37 degrees Celsius in the shaker.
  7. Centrifuged at room temperature for 3 minutes @ 8000 rpm.
  8. Discard 900 micro ml of the super-natured solution and dissolve pellets in remaining 100 micro ml.
  9. These were then spread on LB Agar plates containing 100 micro gram per ml Ampicillin.
  10. Plates were incubated at 37 degrees Celsius overnight.