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Edinburgh/12 September 2008
From 2008.igem.org
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Week 13
Friday 12 September 08
- Analytical double digests of cex (M150) and cex mutant (P96)with EcoRI and PstI, run on Gel 75. (Yan)
- Initially, it was believed that cex mutagenesis had failed. Since no cex-length band was present, we surmised that PstI had cut cex in half at the restriction site we were trying to remove.
- Re-interpretation: We forgot that MABEL PCR results in a linear product with its ends comprising the site of mutagenesis. Digestion of such a product with EcoRI/PstI is meaningless as this generates a pSB1A2 fragment plus two fragments each half the length of cex regardless of whether mutagenesis has occurred. For all we know, P96 might have been successful and we should continue working on it. (AM)
- Repeated double digests of M198, M202~203, M204 for double checking, run on Gel 75. Results indicate failure. (YAN)
- Subplating:
- Colonies from Plates 188 and 189 (dxs+crtE into crtBI) --> Plate 198.
- Colonies from Plates 191 (pSB1A2-bglX) --> Plate 199.
- There were no white colonies and only a few blue colonies to be found on Plates 190 (pSB1A2-bglX), 192 and 193 (both pSB1A2-cenA); blue colonies were present on control Plate 194 but not in expected quantities. These plates shall be checked again on Monday. (AM)
- Maxiprep X22 made from streak 3 on Plate 155, expected to be equivalent to M162 (pSB1A2-dxs+lims+appY). (AM)
