Week from August 24th, to August 30th, 2009

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August, 24th

• Miniprep for B7 (2 samples), B8 (5 samples), A14-3, A16-4 and I714891. Bacterial pellets were stored at -20°C.

• Screening digestion for all the miniprepped samples:
  • B7(E-P)
  • B8(E-P)
  • A14-3(E-P)
  • A16-4(E-P)
  • I714891(E-P)

• Gel run for the digested samples.

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• Gel results:
  • B7 - both screened colonies were negative (~3200bp of vector and ~3200bp of insert).
  • B8 - only B8-5 shows the expected length for ligated plasmid (~3200bp of vector and ~4200bp of insert), but it also shows a single unexpected band...All the other 4 samples were negative (~3200bp of vector and ~3200bp of insert).
  • A14-3 - showed the expected length for ligated plasmid (2079bp of vector and ~2200bp of insert).
  • A16-4 - showed the expected length for ligated plasmid (2079bp of vector and ~1800bp of insert).
  • I714891 - showed the expected length for ligated plasmid (~2700bp of vector and ~700bp of insert).

• We sent purified DNA of:
  • B8-5
  • A14-3
  • A16-4
• to BMR Genomics for sequencing.

August, 25th

August, 26th

• We transformed B5new, A17 and A18 overnight ligations in TOP10. We plated transformed bacteria on LB agar plates + Kan (for B5new and A18) or + Amp (for A17). We incubated B5new plate at 37°C in the morning.

• After about 10 hours we picked 8 colonies from B5new plate and infected 4 ml of LB + Kan to grow overnight cultures (37°C, 220 rpm) for screening.

• A17 and A18 plates were incubated overnight at 37°C.

• We received sequencing results for:
  • A15-1 - sequence ok! (long part) (actually it is not consistent with the Registry, but it is consistent with iGEM 2009 QC, in which there are some problems with K112808, even if the part should work)
  • A15-3 - sequence ok! (long part) (actually it is not consistent with the Registry, but it is consistent with iGEM 2009 QC, in which there are some problems with K112808, even if the part should work)
  • A11-2 - deletion of 20bp in lacI gene...
  • A11-3 - sequence ok!
  • B5-3 - sequence ok! (long part)
  • B6-3 - sequence ok! (long part)
  • B3-5 - sequence ok!

• COMMENTS:
  • we used A11-2 and A15-2 for A14 and A18 ligations...so A14 had to be repeated using A11-3, while we will send A15-2 to BMR for sequencing soon (we forgot to do it!).
  • B3 native stock sequence was confirmed! but we still don't know why we see two unexpected bands after B3 digestions...
  • B5-3 and B6-3 sequences were confirmed! but we still don't know why we see two unexpected bands after B5 and B6 digestions...

August, 27th

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August, 28th

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