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Team:Hawaii/Notebook/2008-08- 7
From 2008.igem.org
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===Verification of transformants=== | ===Verification of transformants=== | ||
| - | [[Image:080708colonyPCR.jpg|right|thumb|500px|EtBr stained 2.5% agarose gel ran at 95V for | + | [[Image:080708colonyPCR.jpg|right|thumb|500px|EtBr stained 2.5% agarose gel ran at 95V for 75 min. Ten microliters of the PCR reaction were loaded into each well.]] |
:<strong>Grace</strong> | :<strong>Grace</strong> | ||
| Line 38: | Line 38: | ||
:* Colony PCR of transformants | :* Colony PCR of transformants | ||
| - | :* Ran on EtBr stained 2.5% agarose gel at 95V for --- | + | :* Ran on EtBr stained 2.5% agarose gel at 95V for 75 min. |
| + | ::* Looks like nir+rbs and I14032+rbs worked. YAY :o). Will prep for sequencing tomorrow. | ||
| + | ::* GFP, GFPf, J33207 don't look like they went in. Will sequence to confirm anyway. | ||
| + | |||
| + | |||
| + | ===Plasmid Prep=== | ||
| + | |||
| + | <strong>Margaret</strong> | ||
| + | [[Image:Plasmid_prep_psmc121.jpg|right|thumb|150px|Plasmid Prep of pSMC121.]] | ||
| + | :*Re-did yesterday's plasmid prep of pSMC121 because I don't believe the band was big enough. I e-mailed Dr. Callahan to get the exact size, but I know, because it contains the omega interposon, that it is at least 2kb. | ||
| + | |||
| + | ===Extraction from Gel=== | ||
| + | |||
| + | <strong>Margaret</strong> | ||
| + | |||
| + | :*extracted 3 bands of 50ul reaction for rep and oriV first by cutting out using long wave UV for visualization. | ||
| + | :* Purification from gel with Qiagen Minelute gel purification kit. | ||
| + | |||
| + | ===PCR=== | ||
| + | [[Image:Large_scale_aada_p1.jpg|right|thumb|150px|Large scale PCR for amplification of P1 lytic and aadA region.]] | ||
| + | <strong>Margaret</strong> | ||
| + | |||
| + | :*Large-scale PCR amplification of aadA (it was lost in a gel cutting mishap) and p1 lytic. | ||
| + | |||
= Discussion = | = Discussion = | ||
Latest revision as of 01:27, 16 August 2008
| Projects | Events | Resources | ||
|---|---|---|---|---|
| Sponsors | Experiments | Milestones | Protocols | |
| Notebook (t) | Meetings (t) |
Things we did today
Wetlab work
Plasmid prep
- Krystle
- Finished plasmid prep of BB-pRL1383a, nir, GFPf from yesterday
Cyanobacteria seed stocks
- Krystle
- Reinoculated liquid and solid media seed stocks
Verification of transformants
- Grace
| Construct | Colony forming units |
|---|---|
| nir + B0030 (rbs) | 2 + 1 cluster of colonies |
| plac (I14032) + B0030 (rbs) | 37 |
| GFP (E0040) + B0015 (tt) | 8 |
| GFPf + B0015 (tt) | 4 |
| lac device (J33207) + B0015 (tt) | 56 |
- Colony PCR of transformants
- Ran on EtBr stained 2.5% agarose gel at 95V for 75 min.
- Looks like nir+rbs and I14032+rbs worked. YAY :o). Will prep for sequencing tomorrow.
- GFP, GFPf, J33207 don't look like they went in. Will sequence to confirm anyway.
Plasmid Prep
Margaret
- Re-did yesterday's plasmid prep of pSMC121 because I don't believe the band was big enough. I e-mailed Dr. Callahan to get the exact size, but I know, because it contains the omega interposon, that it is at least 2kb.
Extraction from Gel
Margaret
- extracted 3 bands of 50ul reaction for rep and oriV first by cutting out using long wave UV for visualization.
- Purification from gel with Qiagen Minelute gel purification kit.
PCR
Margaret
- Large-scale PCR amplification of aadA (it was lost in a gel cutting mishap) and p1 lytic.
Discussion
Quote of the Day
Gernot: "So would you eat the E. coli to save them if we lose the -80C in like a war?"
Loren: "Well, I'll eat them, but you'll be collecting them."
[http://manoa.hawaii.edu/ 
][http://manoa.hawaii.edu/ovcrge/ 
][http://www.ctahr.hawaii.edu 
]
