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Team:Hawaii/Notebook/2008-08- 6
From 2008.igem.org
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| - | [[Image:080608 colony pcr.jpg|right|thumb|200px|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===[[Image:080608 colony pcr.jpg|right|thumb|200px|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.]] |
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:<strong> Grace</strong> | :<strong> Grace</strong> | ||
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:* Reply from CORE Hawaii | :* Reply from CORE Hawaii | ||
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Revision as of 07:19, 7 August 2008
| Projects | Events | Resources | ||
|---|---|---|---|---|
| Sponsors | Experiments | Milestones | Protocols | |
| Notebook (t) | Meetings (t) |
Things we did today
Wetlab work
===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
- made large quantities of aadA, rep, and oriV. Please refer to The experiment write-up for more details.
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
[http://manoa.hawaii.edu/ 
][http://manoa.hawaii.edu/ovcrge/ 
][http://www.ctahr.hawaii.edu 
]
