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Team:Hawaii/Notebook/2008-08- 8
From 2008.igem.org
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:**gfp, gfp''fusion'', and B0015 digested with XbaI and PstI | :**gfp, gfp''fusion'', and B0015 digested with XbaI and PstI | ||
:* Gel Purified restriction digest | :* Gel Purified restriction digest | ||
| - | : | + | :* 2% agarose gel ran at 60 volts for 1.5 hours |
| - | : | + | :::: 40ul of the total restriction digest loaded into each well |
:** gfp, gfp''fusion'', I14032+B0030, nir+B0030 extracted using Qiagen MiniElute tubes | :** gfp, gfp''fusion'', I14032+B0030, nir+B0030 extracted using Qiagen MiniElute tubes | ||
:** B0015 and J33207 extracted using Qiagen cDNA purification tube | :** B0015 and J33207 extracted using Qiagen cDNA purification tube | ||
| + | [[Image:080808resdig.jpg|right|thumb|200px|EtBr stained 2% agarose gel ran at 60V for 60 min. forty microliters of the restriction digest were loaded into each well.]] | ||
= Discussion = | = Discussion = | ||
Revision as of 03:24, 9 August 2008
| Projects | Events | Resources | ||
|---|---|---|---|---|
| Sponsors | Experiments | Milestones | Protocols | |
| Notebook (t) | Meetings (t) |
Things we did today
Wetlab work
Restreaked nir+rbs and I14032+rbs constructs
- Grace
Plasmid prep (finished up)
- Grace
- Resuspended plasmid preps in 50 μl TE buffer
- Determined DNA concentrations of plasmids
| Plasmid | DNA Concentration |
|---|---|
| nir | 496.3 ng/μl |
| GFPf | 484.8 ng/μl |
| BB-pRL1383a | 508.0 ng/μl |
Prep for sequencing
- Grace
- 25 μl PCR reactions of nir+rbs, I14032+rbs, slr1, slr2, BB-pRL1383a
- Colony PCRs seem to indicate success
- 25 μl PCR reactions of GFP+tt, GFPf+tt, J33207+tt
- Colony PCR indicates no ligation. Picked new colony, sequence to confirm failure.
- PCR of nir, B0015, B0030, B0034 for resequencing (bad reads last time)
- Gel purified all PCR rxns (we still have a problem with contaminant DNA/shadow bands) and desired bands were extracted from gel
- 2% agarose gel ran at 60v for 2 hours
- Determined DNA concentrations via nanodrop spectrometer
- Prepared samples and sent to CORE Hawaii for sequencing
Construct p+r+g and p+r+s
- Krystle
- Restriction Digest
- nir+B0030, I14032+B0030, J33207 digested with SpeI and PstI
- gfp, gfpfusion, and B0015 digested with XbaI and PstI
- Gel Purified restriction digest
- 2% agarose gel ran at 60 volts for 1.5 hours
- 40ul of the total restriction digest loaded into each well
- gfp, gfpfusion, I14032+B0030, nir+B0030 extracted using Qiagen MiniElute tubes
- B0015 and J33207 extracted using Qiagen cDNA purification tube
- Restriction Digest
File:080808resdig.jpg
EtBr stained 2% agarose gel ran at 60V for 60 min. forty microliters of the restriction digest were loaded into each well.
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 
]
