Team:Hawaii/Notebook/2008-08- 6

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Things we did today

Wetlab work

EtBr stained 2% agarose gel ran at 95V for 1 hour. Ten microliters of each colony PCR reaction were loaded into each well. Positive control was a nir colony PCR (previously confirmed) and negative control was water.

Checked transformants from yesterday

Grace
Construct Colony forming units
I14032 (plac) + B0030 (rbs) 3
pnir + B003 (rbs) 0
E0040 (GFP) + B0015 (tt) 1
GFPf + B0015 (tt) 2 + 2 clusters of colonies
  • Colony PCR'd transformants
  • 30 cycles, anneal at 62C, extend for 1 min.
  • Ran on EtBr stained 2.0% agarose gel at 95V for 1 hour
  • None of the transformations were successful :o(

Determined DNA concentrations of purified RE'd PCR products from 8/4

Grace
DNA sample Concentration
nir 17.0 ng/μl
slr1 31.3 ng/μl
slr2 11.8 ng/μl
pilA 13.0 ng/μl
GFP 12.0 ng/μl
GFPf 12.2 ng/μl
E0240 8.6 ng/μl
I14032 16.8 ng/μl

Prepared PCR'd nir and J33207 (from yesterday) for transformation/ligation

Grace
  • Ran PCR products on EtBr stained 2.0% agarose gel at 60V for 100 min.
  • nir band at 330bp confirmed
  • J33207 = 4 bands (1.5kb, 2.5kb, 3.2kb, 10kb), none of which are correct
  • [http://www.partsregistry.org partsregistry] says J33207 DNA is inconsistent
  • Extracted nir and 1.5kb J33207 band from gel
  • RE digested in 50 μl rxns with EcoRI and SpeI in NEBuffer 2
  • Larger rxn volume may improve digest efficiency
  • Incubated at 37C for 2.5 hours
  • RE digested 10 μl J33207 plasmid prep with EcoRI and SpeI in NEBuffer 2
  • To confirm plasmid prep; if good, will use for ligation rxn
  • Incubated at 37C for 2 hours
  • Ran RE digests on EtBr stained 2.0% agarose gel at 72V for 90 min.

Made LB+amp100 plates

Grace and Margaret

Plasmid Prep

Margaret
  • pSMC121 was plasmid prepped today


PCR

Margaret
PCR amplification of large quantities of rep, aada, and oriV.

Drylab work

Sequencing

Grace
  • Reply from CORE Hawaii

Discussion

Quote of the Day

History is the only laboratory we have in which to test the consequences of thought. - Étienne Gilson


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