IGEM:Virginia/2012/Notebook/Genetically engineered bacteriophage for diagnosis of whooping cough/2012/10/16

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• Isolated and digested T7 phage genome but suffered from significant degradation due to time.  Therefore, the genome was prepared again using M9LB media and M9 salt media as per protocol.
• Isolated and digested T7 phage genome but suffered from significant degradation due to time.  Therefore, the genome was prepared again using M9LB media and M9 salt media as per protocol.
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• The SynCG BioBrick was digested with only XbaI for 45 minutes and then EcoRI-HF for an additional 15 minutes.  In order to determine if self-ligation is a deciding factor in our experiments, this BioBrick was then transformed with the T7 genome: some samples that had their phosphate groups on the 5’ ends removed with calf-intestinal alkaline phosphatase (CIAP) and some that had not.  The DH5α transformations were plated overnight while the BL21 transformations were analyzed with plague assays.  The former produced only some colonies, likely pointing towards T7 contamination while the plague assays grew too long because they were allowed to incubate overnight.  The plague assays were redone and only allowed to incubate for 3 hours.  The clearings were not characteristic of T7 and there were no identifiable plaques.
• The SynCG BioBrick was digested with only XbaI for 45 minutes and then EcoRI-HF for an additional 15 minutes.  In order to determine if self-ligation is a deciding factor in our experiments, this BioBrick was then transformed with the T7 genome: some samples that had their phosphate groups on the 5’ ends removed with calf-intestinal alkaline phosphatase (CIAP) and some that had not.  The DH5α transformations were plated overnight while the BL21 transformations were analyzed with plague assays.  The former produced only some colonies, likely pointing towards T7 contamination while the plague assays grew too long because they were allowed to incubate overnight.  The plague assays were redone and only allowed to incubate for 3 hours.  The clearings were not characteristic of T7 and there were no identifiable plaques.

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• Isolated and digested T7 phage genome but suffered from significant degradation due to time. Therefore, the genome was prepared again using M9LB media and M9 salt media as per protocol.
• The SynCG BioBrick was digested with only XbaI for 45 minutes and then EcoRI-HF for an additional 15 minutes. In order to determine if self-ligation is a deciding factor in our experiments, this BioBrick was then transformed with the T7 genome: some samples that had their phosphate groups on the 5’ ends removed with calf-intestinal alkaline phosphatase (CIAP) and some that had not. The DH5α transformations were plated overnight while the BL21 transformations were analyzed with plague assays. The former produced only some colonies, likely pointing towards T7 contamination while the plague assays grew too long because they were allowed to incubate overnight. The plague assays were redone and only allowed to incubate for 3 hours. The clearings were not characteristic of T7 and there were no identifiable plaques.



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