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Edinburgh/24 July 2008
From 2008.igem.org
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Week 6
Thursday 24 July 08
- Purified PCR products P28 (rbs+crtB) and P29 (rbs+crtI). (OG)
- Double digestions using XbaI/PstI (buffer H) to add P12 (rbs+dxs), P15 (rbs+appY), P28 (rbs+crtB) and P29 (rbs+crtI) to Edinbrick1. (Yan, OG)
- Double digestion using SacI/SpeI (BufferE) to add P31 (PzntA) to Edinbrick1. (Yan, OG)
- Purified genomic DNA from C. fimi culture (made from plate 37, incubated in 25ml bottle at 30C for 48 hours with shaking). Used Promega Wizard Genomic DNA Purification Kit, lysozyme and RNAse+EDTA (as replacement for proprietary RNAse Solution, of which none was left). Final solution left overnight in refrigerator at 4°C. (AM)
- Plated C. fimi culture from plate 37...
- Incubated in 25ml bottle for 48 hours at 30°C with shaking ... to Plate 48.
- Incubated in 50ml bottle for 24 hours at 30°C with shaking ... to Plate 49.
- Incubated in 250ml flask for 24 hours at 30°C with shaking ... to Plate 50. (AM)
- PCR of cenA (P32), cenB (P33), cenC (P34), cex (P35), annealing 1 min 55°C, extension 90 sec 70°C. Run on Gel 24 - unsuccessful. (AM)
