My Notes II

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07 August 2008 (Th)

  • Picked colonies for sca2-7; streaked on CK plate, screened on Amp. Growing overnight in CK media
  • mini meeting - new methods enforced due to assembly status:

Assembly Status: 

All transfers completed successfully, EXCEPT sca2 (rbs1-A) and sca3 (fimH)

Layer one assemblies completed:
sca21-{<phoA!}.{b1006}
sca22-{<AP!}.{b1006}
sca23-{<HA!}.{b1006}
sca24-{<myc!}.{b1006}
sca25-{<FLAG!}.{b1006}

None of Layer 2 or 3.
  • Decided to shrink old tree and use new methods to finish the tags: attach my {<tag!}.{b1006} parts to {FimH}.{phoA!}
    • Take existing {FimH}.{PhoA!} (aka. pBca1102-Bca9194), set up PCR using ca998 oligo and mea37 (<phoA> reverse oligo) to remove stop codon
    • At first, the PCR product came out mysteriously large. We tried 3 times and the same thing occurred.

It was not until 11:00pm, that figured that I was using the wrong oligo ... we set up the 4th PCR then - with the correct oligo.


  • also amplified Bca9194 for personal stock, since we were running low - grew on amp plate AND amp media
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