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Team:Hawaii/Notebook/2008-10- 6
From 2008.igem.org
| Projects | Events | Resources | ||
|---|---|---|---|---|
| Sponsors | Experiments | Milestones | Protocols | |
| Notebook (t) | Meetings (t) |
Things we did today
Wetlab work
Construction of secretion device (cont.)
- Grace
- PCR'd nir, nir+rbs, rbs+GFP+tt#2, plac+rbs, slr1+GFPf
- Verified PCR products on 0.8% agarose gel
- Overnight RE digest:
- EcoRI/SpeI:
- nir
- nir+rbs
- plac+rbs
- XbaI/PstI;
- slr1+GFPf
- pilA+GFPf+tt
- rbs+GFP+tt#2
- HindIII/BamHI:
- J33207
OriV construction
- Margaret
- Ligation
- oriV + pSB1A3 (de-phosphorylated)
- Transformation into DH5-alpha, with amp100 selection
- 10/4 ligation
- 10/6 ligation
- (+) control pUC18
- (-) control no plasmid
- PCR verification
- colonies 5 and 6 were PCR verified
- a gel was run
- oriV1---> band at 400 bp
- oriV2---> no band
- oriV3---> no band
- oriV4---> no band
- oriV5---> band at 700 bp
- oriV6---> band at 1000 bp
- rep 2---> band at 3000 bp
- plac/rbs/rep (colony 8)---> band <3kb (need to re-do this)
plac/rbs/rep and plac/rbs/aada
- Margaret
- plasmid prep of both constructs
- Restriction digest for 2 hours at 37°C, then a gel was run to verify the digest
- rep2 with X,P
- plac/rbs/rep (colony 8) with X,P
- plac/rbs/rep (colony 8) with E,S
- plac/rbs/aada with X,P
- PCR verification of more plac/rbs/rep colonies (14 total) and also colony 8 again to verify that I have what I think I do. (+) was pSB1A3, (-) was water
Drylab Work
Sequencing analysis
- Grace
- Correct:
- rbs+GFP+tt #1, 2
- BBpRL1383a:
- All four sequenced colonies had a G->C single bp substitution at position 229 of part (in CAP binding site)
- Colony 18 confirmed (best quality sequence)
- Colony 1 confirmed (abiguities at 225, 583, 591, 552 of part)
- Colony 3 (abiguities at 530, 513 of part)
- Colony 13 (low quality sequence, but BioBrick MCS is present)
- Incorrect:
- nir+rbs+GFP #4, 7
- nir+rbs+slr1+GFPf #8
- No sequence obtained:
- plac+rbs
- nir+rbs+GFP #1
- nir+rbs+slr1+GFPf #2
- nir+rbs+pilA #17
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 
]
