. As a backup, the running some of the running culture of the Comparator circuit 1 and 2 transformed cells were mini-prepped. After nanodropping, the concentration was found to be:
. As a backup, the running some of the running culture of the Comparator circuit 1 and 2 transformed cells were mini-prepped. After nanodropping, the concentration was found to be:
C1-1a: 733.4 ng/µL
C1-1a: 733.4 ng/µL
+
C1-2a: 1192.6ng/µL
C1-2a: 1192.6ng/µL
+
C1-3a: 1596.9 ng/µL
C1-3a: 1596.9 ng/µL
+
C1-4a: 854.8 ng/µL
C1-4a: 854.8 ng/µL
+
C2-1a: 896 ng/µL
C2-1a: 896 ng/µL
+
C2-2a: 712.5 ng/µL
C2-2a: 712.5 ng/µL
+
C2-3a: 1196.7 ng/µL
C2-3a: 1196.7 ng/µL
+
C2-4a: 811.9 ng/µL
C2-4a: 811.9 ng/µL
. Following this an overnight double restriction digest with ''Eco''R1 and ''Pst''1 was set up. These were set up using: 0.2µl BSA, 2µl Buffer H, 0.5µl EcoR1 and 0.5µl Pst1. As to the DNA, 1µg of DNA was added:
. Following this an overnight double restriction digest with ''Eco''R1 and ''Pst''1 was set up. These were set up using: 0.2µl BSA, 2µl Buffer H, 0.5µl EcoR1 and 0.5µl Pst1. As to the DNA, 1µg of DNA was added:
. As a backup, the running some of the running culture of the Comparator circuit 1 and 2 transformed cells were mini-prepped. After nanodropping, the concentration was found to be:
C1-1a: 733.4 ng/µL
C1-2a: 1192.6ng/µL
C1-3a: 1596.9 ng/µL
C1-4a: 854.8 ng/µL
C2-1a: 896 ng/µL
C2-2a: 712.5 ng/µL
C2-3a: 1196.7 ng/µL
C2-4a: 811.9 ng/µL
. Following this an overnight double restriction digest with EcoR1 and Pst1 was set up. These were set up using: 0.2µl BSA, 2µl Buffer H, 0.5µl EcoR1 and 0.5µl Pst1. As to the DNA, 1µg of DNA was added:
C1-1a: 1.36 µL
C1-2a: 0.84 µL
C1-3a: 0.62 µL
C1-4a: 1.17 µL
C2-1a: 1.12 µL
C2-2a: 1.40 µL
C2-3a: 0.84 µL
C2-4a: 1.23 µL
. Following a low concentration reading on Friday, B-M was miniprepped again. However, this produced no DNA when nanodropped. Therefore, B-M was reinoculated into culture.
. M-B samples were digested with Spe1 and Pst1 to linearise the backbone to ligate a reporter (CFP, or RFP) into it. The small fragment that was released was discarded during gel purification. At the same time CFP and RFP were digested with Pst1 and Xba1 and then gel purified ready for ligation with M-B. The restriction digests involved 0.5µL of each enzyme, 2µL of Buffer B (Spe1 and Pst1)and Buffer H (Pst1 and Xba1), 0.2µL of BSA. Again 1µg of DNA was used. The gel used to separate the fragments was 1% w/v.
. Multiple ligations were set up. MB2, MB3,MB5,MB9,MB10 were set up to ligate with both RFP and CFP. Comparator circuit 1 and 2 (all samples) were set up to be ligated in the iGEM backbone pSB1C3. These were all left overnight.
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