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Team:Paris/31 July 2009
From 2009.igem.org
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===Lab work=== | ===Lab work=== | ||
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DID dying to DH5a and MG4 | DID dying to DH5a and MG4 | ||
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We don't see florescence | We don't see florescence | ||
->do it again but wash the cells before the DID | ->do it again but wash the cells before the DID | ||
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| - | + | <div class="guillaume"> | |
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Miniprep | Miniprep | ||
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Miniprep (Pure Yield Tm Plasmid Miniprep System) for [S7] which contain g3p expression plasmid [[https://2009.igem.org/Team:Paris/Miniprep Protocol]] | Miniprep (Pure Yield Tm Plasmid Miniprep System) for [S7] which contain g3p expression plasmid [[https://2009.igem.org/Team:Paris/Miniprep Protocol]] | ||
</div> | </div> | ||
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Culture ON for glycerol stock | Culture ON for glycerol stock | ||
| - | </div><div | + | </div> |
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*[S4] TB28 LB | *[S4] TB28 LB | ||
*[S5] MG4 LB Kan NaCl 1% | *[S5] MG4 LB Kan NaCl 1% | ||
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*[S8] DH5α with puc19 LB Amp (control for Amp resistance) | *[S8] DH5α with puc19 LB Amp (control for Amp resistance) | ||
</div> | </div> | ||
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Biobrick resuspention | Biobrick resuspention | ||
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*1/6I named 1I6 (BBa_R0040, TetR repressible promoter). Resistant to Ampicilin. | *1/6I named 1I6 (BBa_R0040, TetR repressible promoter). Resistant to Ampicilin. | ||
*1/14N named 1N14 (BBa_10500, pBad/araC). Resistant to Kanamycin. | *1/14N named 1N14 (BBa_10500, pBad/araC). Resistant to Kanamycin. | ||
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*pTW2 (VAMP2-HIS6). Resistant to Ampicilin. | *pTW2 (VAMP2-HIS6). Resistant to Ampicilin. | ||
</div> | </div> | ||
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Transformation of precited Biobrick | Transformation of precited Biobrick | ||
| - | </div><div | + | </div> |
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*250µl of DH5α competent bacteria (by RbCl) | *250µl of DH5α competent bacteria (by RbCl) | ||
*Add 2µl of DNA (Biobrick) | *Add 2µl of DNA (Biobrick) | ||
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*Incubate overnight at 37°C | *Incubate overnight at 37°C | ||
</div> | </div> | ||
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Transformation efficiency (cfu/µg) | Transformation efficiency (cfu/µg) | ||
| - | </div><div | + | </div> |
| + | <div class="experience"> | ||
*cfu/µg = nb colonies/µg DNA | *cfu/µg = nb colonies/µg DNA | ||
*For RbCl, transformation efficiency = 1,2.10<sup>7</sup>cfu/µg | *For RbCl, transformation efficiency = 1,2.10<sup>7</sup>cfu/µg | ||
Revision as of 21:49, 7 August 2009
NoteBook
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Lab work
DID dying to DH5a and MG4
We don't see florescence
->do it again but wash the cells before the DID
Miniprep
Miniprep (Pure Yield Tm Plasmid Miniprep System) for [S7] which contain g3p expression plasmid [Protocol]
Culture ON for glycerol stock
- [S4] TB28 LB
- [S5] MG4 LB Kan NaCl 1%
- [S3] Δnlp1 LB Kan
- [S8] DH5α with puc19 LB Amp (control for Amp resistance)
Biobrick resuspention
- 1/6I named 1I6 (BBa_R0040, TetR repressible promoter). Resistant to Ampicilin.
- 1/14N named 1N14 (BBa_10500, pBad/araC). Resistant to Kanamycin.
- 1/2J named 1J2 (BBa_J32015, PelB leader sequence). Resistant to Kanamycin.
- 1/18C named 1C18 (BBa_J61002, plasmid backbone). Resistant to Ampicilin.
- 1/7C named 1C7 (pSB2K3). Resistant to Kanamycin.
- 2/11P named 2P11 (BBa_I14032, pLac). Resistant to Kanamycin.
- 2/24C named 2C24 (BBa_B0014). Resistant to Kanamycin.
- 2/22P named 2p22 (BBa_K103006, OmpA linker). Resistant to Ampicilin.
- pFP141 (&DeltaN-Syn1A). Resistant to Kanamycin.
- pJM72 (His6-SNAP25). Resistant to Ampicilin.
- pTW2 (VAMP2-HIS6). Resistant to Ampicilin.
Transformation of precited Biobrick
- 250µl of DH5α competent bacteria (by RbCl)
- Add 2µl of DNA (Biobrick)
- Incubation during 20min on ice
- Heat choc 45second at 42°C
- Leave on ice 2min
- Add 1ml of LB preheated at 42°C
- Incubation 1 hour at 37°C under agitation
- Put 100µl of culture under plate
- Centrifuge 2min at 6rpm MAX for 2 min
- Put 100µl of concentrated culture
- Incubate overnight at 37°C
Transformation efficiency (cfu/µg)
- cfu/µg = nb colonies/µg DNA
- For RbCl, transformation efficiency = 1,2.107cfu/µg
To do list
| Matricule | TODO |
| Luc | SNARE Construction |
| Romain | Design oligo Fec A |
| Charlotte | Absent |
| Stoff | Dev / Labs |
| Chris | Global Design / Wiki / Modeling |
| Lisa | Oligo Tol/Pal / Oligo Sequencing / Tol/Pal Protocol verification / Keio / Theme A writting |
| Caroline | Oligo Tol/Pal / Oligo Sequencing / Tol/Pal Protocol verification / Keio / Theme A writting |
| Souf | Tat/Sec system / Design signal peptid |
| Vicard | ClyA protocol |
| Pierre | Modeling/SNARE |
| Sylvain | Absent |
| Guillaume | Labs / Protocol for g3p control / Thematique A / Sequencage OmpA-linker-G3P oligo |
