840:153g:Projects/project22/2012/11/29: Difference between revisions

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* 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).
* 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.
* 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:
 
[[Image:29.11.12_small.png‎]]
 
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.
 




Revision as of 17:14, 29 November 2012

Prodigiosin Production by E. Coli <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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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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