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Edinburgh/1 July 2008
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Week 3
Tuesday 1 July 08
Gel 3: minipreps M2, M3, M4 and M6 (pSB1A2+dxs clones) digested with EcoRI alone; lanes 5 and 6, 2.5μl of ligations L1 and L2. Result: all 4 minipreps now give a 4.2kb band (plus the same 3.2kb 'ghost' band as before) consistent with pSB1A2 carrying a 2kb insert. Would be nice to confirm identity using HindIII (2 internal sites). Both ligations show clear signs of insert and vector bands (oddly, the dxs insert band overlaps the pSB1A2 vector band whereas the appY insert band overlaps the lacZ insert excised from Edinbrick1). However, no ligated bands are visible. Thus the DNA purification is fine; if there was a a problem, it is with the ligase or ligase buffer.
Gel 4: Minipreps M7 to M12 (supposed to be glgC in Babel vectors) digested with EcoRI and PstI to excise the inserts. M10 and M11 have a single 3kb band consistent with vector, but no insert band at 1.2kb. M7, M8, M9 and M12 all show a single band at about 2.4kb which is not consistent with Babel vectors if properly digested. Unless the digests totally failed, none of these plasmids would seem to contain glgC.
- BUT WAIT: Turns out gel 4 has undigested plasmid DNA rather than the EcoRI/PstI digests. These were run on Gel 5. Since glgC is about 1295bp with internal EcoRI sites at +570 and +1067, we would expect to see a vector band at 3kb and the insert cut into bands of around 570bp, 567bp and 228bp. This is precisely what we see in the case of M10 and M11. We can therefore conclude that M10 and M11 both represent the Babel2+glgC BioBrick. This is not sufficient to determine the orientation, but a single EcoRI digest of these two clones should be sufficient to establish that. Even if both are in the reverse orientation, they can still be used as templates for the three mutagenesis step which will be required to produce the final BioBrick.
- Also attempted HindIII digests of M2, M3, M4 and M6 to confirm that the insert is really dxs, despite knowing that the HindIII stock expired in 2002. Results are shown on Gel 6 lanes 1 to 4. In all cases a 0.6kb band was excised, consistent with dxs insert. It therefore seems likely that M2, M3, M4 and M6 all represent pSB1A2+dxs coding sequence BioBrick.
- Also did fusion PCR for the BABEL1+glgC and BABEL2+glgC constructs, using 1μl of L3 and L4 as templates (P4 and P5). KOD was used, with primers stdvectf1 and glgCr1, annealing at 65°C and extending for 110s. Results are shown on Gel 6 lanes 5 and 6. P4 (BABEL1) has a strong band the right size and would probably work if self-ligated, but P5 (BABEL2) shows strong bands at 2 and 3 kb, as CK is also seeing. I suspect that primer stdvectf1 does not work well with BABEL2 - possibly mis-anneals to the other end of the vector. In any event, we can delay the decision as to whether or not to carry on with these until we know the results from M10 and M11.
