Edinburgh/4 July 2008


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Friday 4 July 08

 * Repeat appY transformation has failed: no colonies on plates 15 and 16. It seems unlikely that there is a major problem with the competent cells since other transformations have worked. Other than this, the only way to get no colonies (not even recircularized vector) is if something went seriously wrong in the original digest.
 * Mutagenic primers for glgC arrived. Prepared 500μM stock solutions and 10μM working solutions. Tried PCR with both primer pairs using M11 (0.5μl) as template, just to check that the primers are OK. Annealing was at 57°C (lowest melting temperature of a primer) and extension for 110s using KOD (PCR reactions P6 and P7). Products (5μl) were run on Gel 8. P6 (M1) shows a single rather faint band at 4.2kb; P7 (M2) shows multiple bands, with the 4.2kb band strongest. Probably both pairs are fine for MABEL. Now we just need to wait for the sequencing results on Monday.
 * Also ran the other 5μl left from the religated L2 on gel 8. No DNA was visible. Did another digest (30μl total volume with 2μl each P2 and Edinbrick1, using Buffer E with EcoRI and SpeI, digesting at 37°C for about 5 hours, mainly because I forgot about it). Purified and ligated: Ligation L5.


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