IGEM:Virginia/2012/Notebook/Genetically engineered bacteriophage for diagnosis of whooping cough/2012/08/19

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Week of 8/19/12

• Digested T7 genome with SclI. No bands were present in the gel lanes containing the T7 genome. However, when the T7 genome that had been subjected to the same conditions as the digest, sans the addition of SclI enzyme, a smear appeared. Mostly of the smear was concentrated below the 100 bp marker, indicating that the genome was fragmented into very small pieces, either before or during the 4 hour long digestion at 50°C. This is odd as E. Coli (BL21) has been transformed before with the genome and produced intact phage. The genome, therefore, must have been intact for that experiment. Therefore, a gel extraction was run with the T7 genomne directly from the stock tube in the freezer using the QIAEX II kit. However, the kit failed as the solutions were vortexed too haed and too much QX1 buffer was added, resulting in failure.
• Digested pSB1C3 backbone with two combinations of restriction enzymes: EcoRI-HF and SpeI-HF (E+S) as well as EcoTI-HF and PstI-HF (E+P).
• Grew up 5mL DH5α and added 300 µL of 5 mg/µL of kanamycin. After 5 hours, 20 µL of IPTG to 10 mL DH5α and again after a further 14 hours.
• T7 phage was amplified again in DH5α over night; after lysing the cells, the T7 genome was isolated using QIAquick kit. However, the kit needed to be performed twice as the first isolation resulted in co-purification.
• Pregnancy test experiment was repeated with DH5α cells that had been transformed with hCG and induced for hours. The samples which were either doubled or quadrupled in their concentration of cells were then denatured with either a 5 mM DTT solution, a 0.5 mM DTT solution, a 0.05 mM DTT solution, or a 0 mM DTT solution. Samples of each of these either were then subjected to different renaturing solution concentrations of either 6.4 mM : 3.6 mM cysteamine to cystamine or 4.8 mM : 4.4 mM cysteamine to cystamine creating a total of 16 pregnancy tests. All results were negative.