840:153g:Projects/project22/2012/11/29

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Transformation

  • Before break, we began the process of transformation using the pGEM-T Easy Vector kit. On Tuesday we resumed transformation and transformed our PCR product in DH5alpha cells and grew them on ampicillin plates.
  • Plate Results:
  3Amp: 2 colonies (only one was present at the time of picking)
  2Amp: 0 colonies
  1Amp: 0 colonies
  +Amp: 20 colonies
  -Amp: 0 colonies
  -NoAmp: plate completely covered.
  • Since out of all of our sample plates, plate 3 was the only to produce a single colony, it was picked and incubated in 5 mL of luria broth and 5 ul of ampicillin at 37 degrees Celsius and 220 rpm. Growth was observed in all of the test tubes(1-5 positive controls and Ampicillin plate 3 colony).
  • Next, we used the GeneJet Plasmid Miniprep Kit (Lot: 00038294) to purify the plasmid, and ran gel electrophoresis on the product. Our results are as follows:

The top band on the ladder (first lane) represents 3000 b.p. As you can see, we have a bright band above this line (lane 2) which corresponds to the size of our T-vector (~3200 b.p.). We do not, however, see a bright line at ~1500 b.p. which we would expect with successful transformation of our gene (pigI is 1473 b.p.). To confirm our suspicions, we will send off our DNA product for sequencing.


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