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My Notes II
From 2008.igem.org
Contents |
10 August 2008 (Su)
- Came into lab today!!!
- Moved sca4-sca7 into Lefty cells instead of righty. (already in AC vector, still useful)
- Redid everything from yesterday, only using AKL 41 as the Aron part.
09 August 2008 (S)
Busy…very busy…
- Miniprepped 4 colonies of fimH;
- Simultaneously did 3 different types of digestion protocols (with 10 tubes): 1.)mapping 2.)digesting vectors, and 3.)digesting Aron’s parts
- Digestion mapped all 4 miniprepped fimH's. All had different band sizes… sent colony 3 for sequencing.
- Finished up with 16 and 19, each with my parts sca22-25
= total 8 things.
- Left before transformation and plating, but found out in evening that 16 and 19 parts were unnecessary, since Aron had an even more complete part (with pBad promoter)… didn’t plate.
08 August 2008 (F)
- Sca2: both diluted colonies co transformed -- no miniprepping...
- FimH (SC43) came back bad read
- Took fimH from Jin’s Amp plate (same day as re-amplified fimH), picked 4 colonies and grew in LB Amp-Checked plate and culture for amplifying Bca9194 - nothing grew.
- Ran a gel in the morning at 9 a.m. -- gel turned out weird: 2 bands - one at 4000 and other at 650-750ish bp both were wrong… (size was supposed to be 1610 bp)
- talked with Chris and Jin. Stopped using questionable Bca9194; used Aron’s parts - AKL16, 17, and 19 - as PCR template instead.
- Ran gel to check Aron’s 16, 17, and 19 -- used PCR product of AKL16 and AKL19
- Did a zymo for AKL 16 and AKL 19.
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
