From 2008.igem.org
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
Drylab work
Sequencing
- Grace
-
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 
]
"
