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Team:Hawaii/Notebook/2008-09-25
From 2008.igem.org
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::*oriT + J33207(de-phosphorylated) colony 1 | ::*oriT + J33207(de-phosphorylated) colony 1 | ||
::*5 additional colonies of oriTr transformation were PCR'ed | ::*5 additional colonies of oriTr transformation were PCR'ed | ||
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| + | ===Construction of secretion device and redo of BB-pRL1383a (cont.)=== | ||
| + | :<strong> Grace</strong> | ||
| + | [[Image:092508REdigest.jpg|right|thumb|300px|EtBr stained 2% agarose gels ran at 60V for 1.5 hours and 2 hours, respectively. Thirty microliters of RE digested products were loaded into each well.]] | ||
| + | :* Ran RE digests from last night on gel | ||
| + | ::* nrsg8 and nrg4 did not cut. Implies insert wrong? | ||
| + | :* Extracted bands from gel | ||
| + | :* Ligated: | ||
| + | ::* 3A using pSB1A3: | ||
| + | :::* nrg7 + tt | ||
| + | :::* rgt1 + nir | ||
| + | :::* rgt1 + plac | ||
| + | :::* rgt2 + nir | ||
| + | :::* rgt2 + plac | ||
| + | :::* pSB1A3 (control) | ||
| + | :* pRL1383a + J33207 | ||
| + | ::* pRL1383a to self (control) | ||
| + | :* Transformed into DH5α using 5 μl ligation reaction; also transformed J33207 and pRL1383a plasmids as control) | ||
= Discussion = | = Discussion = | ||
Latest revision as of 23:51, 25 September 2008
| Projects | Events | Resources | ||
|---|---|---|---|---|
| Sponsors | Experiments | Milestones | Protocols | |
| Notebook (t) | Meetings (t) |
Things we did today
Wetlab work
Verification of Ligation
- Margaret
- The plac/rbs+rep and oriTr(BB) transformations resulted in 0 colonies with the correct insert, so the remainder of the ligation reaction from 9/16 was digested with XbaI and run on a gel.
Sequencing & Colony PCR
- Margaret
- PCR
- oriV colonies (3,4,6,8,9,10,11)
- aadA(BB) colonies (4,5,7,8)
- aadA(pRL) colonies (1,2)
- oriT + J33207 (1,2,4)
- oriT + J33207(de-phosphorylated) colony 1
- 5 additional colonies of oriTr transformation were PCR'ed
Construction of secretion device and redo of BB-pRL1383a (cont.)
- Grace
- Ran RE digests from last night on gel
- nrsg8 and nrg4 did not cut. Implies insert wrong?
- Extracted bands from gel
- Ligated:
- 3A using pSB1A3:
- nrg7 + tt
- rgt1 + nir
- rgt1 + plac
- rgt2 + nir
- rgt2 + plac
- pSB1A3 (control)
- pRL1383a + J33207
- pRL1383a to self (control)
- Transformed into DH5α using 5 μl ligation reaction; also transformed J33207 and pRL1383a plasmids as control)
Discussion
Quote of the Day
History is the only laboratory we have in which to test the consequences of thought. - Étienne Gilson
