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Team:NRP-UEA-Norwich/Week10
From 2012.igem.org
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Contents |
Everything has really come together this week. In general everything ran extremely smoothly. It is just as a wise man said 'Everything comes together in the final few weeks'. We have successful ligations from BM and MB with fluorescent proteins: CFP and RFP which will allow us to carry out many characterisation experiments next week.
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 and Comparator circuit 1 and 2 (all samples) were set up to be ligated in the iGEM backbone pSB1C3. These were all left overnight.
The ligations (MB with CFP/RFP and Comparator Circuit 1 and 2 with pSB1C3) from the night before were run on a 1% w/v gel for an hour and the fragments were cut out. These gel slices were purified. The purified DNA, was then transformed into Bioline Alpha Select Gold Standard Competence cells. The plates were left overnight in an incubator at 37 degrees Celsius.
Samples of B-M were miniprepped and nanodropped. The concentrations were found to all be lower than 45ng/L. Furthermore, the 260/230 readings were all negative.
A B-M overnight culture was miniprepped by Russell, and nanodropped. The concentration was found to be: 199 ng/µL. This was then digested with Spe1 and Pst1. Again 1µg of DNA, 0.5µL of each of Spe1 and Pst1, 0.2µL BSA and 2µL Buffer B. Following the digestion, this was then run on a 1% w/v agarose gel for an hour and the large fragment purified. Multiple overnight ligations were then set up for BM1 and BM4 with RFP and CFP. These were left in the dark.
All the plates which showed growth from the previous days experiments were inoculated into 5ml LB cultures containing the relevant antibiotics. Furthermore, as back up, Comparator circuit 1 and 2 was transformed again and plated. These plates were grown overnight at 37 degrees Celsius.
Inoculations were made of BM's and MB2 to CFP and RFP. These were inoculated into many 5ml LB cultures with chloramphenicol added. We also Transformed B-M and grew it overnight at 37 degrees Celsius.
The MB2 attached to RFP and CFP were transformed and plated. These plates were incubated over the weekend. We then re-inoculated BM's and MB2 ligated to CFP and RFP.
As this is our final day in the teaching labs, we did a thorough lab cleanup session in preparation to move lab.
