"
Team:Hawaii/Notebook/2008-10- 9
From 2008.igem.org
(Difference between revisions)
(→Triparental conjugtion) |
(margaret) |
||
| Line 35: | Line 35: | ||
:* Restreaked original colonies #1, 3, 13, 18 on freshly made LB+sp<sub>100</sub>+sm<sub>100</sub> | :* Restreaked original colonies #1, 3, 13, 18 on freshly made LB+sp<sub>100</sub>+sm<sub>100</sub> | ||
::* Original colonies scraped w/ loop. Hopefully there's something there. | ::* Original colonies scraped w/ loop. Hopefully there's something there. | ||
| + | |||
| + | ===Construction of plasmid=== | ||
| + | :<strong>margaret</strong> | ||
| + | [[Image:rep_aadA_purification_10_9.jpg|right|thumb|300px|plac/rbs/rep5 (E,S), plac/rbs/rep7 (E,S), plac/rbs/aada (ap32 E,S), plac/rbs/aada ap2 cut with X,P.]] | ||
| + | :* Gel purification of re-digest | ||
| + | ::*plac/rbs/aada (ap32 was cut with E,S), ap2 did not cut correctly | ||
| + | ::*plac/rbs/rep (7 was cut with E,S), rep5 did not cut correctly | ||
| + | :*Ligation, transformation (in DH5 alpha): | ||
| + | ::*oriT + plac/rbs/aada + pSB1A3 (dephosphorylated) | ||
| + | ::*oriV + plac/rbs/rep + pSB1A3 (dephosphorylated) | ||
| + | ::pSB1A3 (dephosphorylated) + self | ||
| + | ::*(+) control for transformation- pUC18 | ||
| + | ::*(-) control for transformation- no plasmid | ||
| + | |||
| + | ===OriV back-ups=== | ||
| + | :<strong>margaret</strong> | ||
| + | :* two colonies were of correct size from yesterday's transformation, so i did a plasmid prep of them and I will store them in -20 C in TE | ||
| + | |||
= Discussion = | = Discussion = | ||
Revision as of 06:47, 10 October 2008
| Projects | Events | Resources | ||
|---|---|---|---|---|
| Sponsors | Experiments | Milestones | Protocols | |
| Notebook (t) | Meetings (t) |
Things we did today
Wetlab work
Construction of secretion device (cont.)
- Grace
File:100908REdigest.jpg
EtBr stained 4% agarose gel ran at 60V for 3.5 hours. Twenty microliters of RE digested product were loaded into each well.
- Ran RE digests on gel
- Extracted bands from gel
- Ligated:
- J33207 and pRL1383a
- nir and rbs+GFPf+tt #1 and pSB1A3
- plac and rbs+GFPf+tt #1 and pSB1A3
- nir+rbs and slr1+GFPf and pSB1A3
- nir+rbs and pilA+GFPf+tt and pSB1A3
- Used 5 μl ligation reaction to transform DH5α
Sequencing
- Grace
- Prepared and sent samples in for sequencing:
- pilA+GFPf+tt #21 (KS)
- nir+rbs+slr1+GFPf #15 (10/2 transformation)
- slr1+GFPf+tt (10/2 transformation)
- nir+rbs+slr1+GFPf #6 (10/8 transformation)
- plac+rbs+pilA+GFPf+tt #5, 7, 11 (10/8 transformation)
Triparental conjugtion
- Grace
- BBpRL #1, 18 cultures did not grow
- PCR of colonies (on Xgal plate) showed no insert present (?!?)
- PCR of colonies from non-Xgal plate) showed no insert present (?!?)
- Faint band for colonies #1, 13 at 400bp ???
- Restreaked original colonies #1, 3, 13, 18 on freshly made LB+sp100+sm100
- Original colonies scraped w/ loop. Hopefully there's something there.
Construction of plasmid
- margaret
File:Rep aadA purification 10 9.jpg
plac/rbs/rep5 (E,S), plac/rbs/rep7 (E,S), plac/rbs/aada (ap32 E,S), plac/rbs/aada ap2 cut with X,P.
- Gel purification of re-digest
- plac/rbs/aada (ap32 was cut with E,S), ap2 did not cut correctly
- plac/rbs/rep (7 was cut with E,S), rep5 did not cut correctly
- Ligation, transformation (in DH5 alpha):
- oriT + plac/rbs/aada + pSB1A3 (dephosphorylated)
- oriV + plac/rbs/rep + pSB1A3 (dephosphorylated)
- pSB1A3 (dephosphorylated) + self
- (+) control for transformation- pUC18
- (-) control for transformation- no plasmid
OriV back-ups
- margaret
- two colonies were of correct size from yesterday's transformation, so i did a plasmid prep of them and I will store them in -20 C in TE
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 
]
