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Edinburgh/25 July 2008
From 2008.igem.org
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Week 6
Friday 25 July 08
- Made maxiprep X4 (pSB1A2+crtB as M36; BBa_K118002) and X5 (pSB1A2+crtI as M42; BBa_K118003) (AH, XH)
- Made ligations:
- L19: P12 (rbs+dxs) into Edinbrick1 to make pSB1A2+rbs+dxs
- L20: P15 (rbs+appY) into Edinbrick1 to make pSB1A2+rbs+appY
- L21: P28 (rbs+crtB) into Edinbrick1 to make pSB1A2+rbs+crtB
- L22: P29 (rbs+crtI) into Edinbrick1 to make pSB1A2+rbs+crtI
- L23: P31 (PzntA) into Edinbrick1 to make (pSB1A2+PzntA). (Yan, OG)
- C. fimi Plate 48 appears to be contaminated (mix of opaque and clear colonies). The same culture was used for genomic DNA purification yesterday. This preparation is therefore inadequate for sequencing purpose but might be worth using for PCR. (AM)
- No growth of C. fimi on Plates 49 and 50, but C. fimi takes a relatively long time to form colonies so we should check again on Monday. (AM)
- PCR P36 of M43 (glgC-mut1,2) to excise the gene from BABEL and rectify its orientation and P37 of crtY from P. ananatis (Plate 17) cell suspension - repeat of P27. (AM)
- P36 and P37 run on Gel 25. P36 tentatively successful (larger than desirable smear in the region of interest), P37 failed. (AM)
- Also did preparations to transfer crtE from BABEL2 vector to pSB1A2. This is necessary, because we are supposed to use Registry standard vectors for all BioBricks we make (in fact, we should even stop using pSB1A2 and move to pSB1A3). So digested the BABEL2+crtE clone (that Douglas Leslie made) with EcoRI/PstI, and also Edinbrick1 - in both cases, 3μl in 20μl total volume. Ran both on a gel and excised the appropriate bands, eluted each to 10μl EB and set up a ligation (L24) using 4μl of each in 10μl total volume. Ligated at 16°C. (CF)
