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Team:Paris/12 August 2009
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===Lab work=== | ===Lab work=== | ||
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<div class="experience"> | <div class="experience"> | ||
| - | *Purification of A10(ClyA) (using 1% agarose during 25 min) | + | *Purification of A10(ClyA) (PCR at 55° during 30sec) (using 1% agarose during 25 min) |
*A10=[https://2009.igem.org/Team:Paris/Freezer_PCR_products#Freezer#A10 A10] 987pb | *A10=[https://2009.igem.org/Team:Paris/Freezer_PCR_products#Freezer#A10 A10] 987pb | ||
<center>[[Image:ClyA_pcr2.JPG]]</center> | <center>[[Image:ClyA_pcr2.JPG]]</center> | ||
| Line 92: | Line 81: | ||
<center>[[Image:ClyA_purif.JPG]]</center> | <center>[[Image:ClyA_purif.JPG]]</center> | ||
*Purification of A10 work | *Purification of A10 work | ||
| + | </div> | ||
| + | |||
| + | <div class="sylvain"> | ||
| + | PCR Gel verification | ||
| + | </div> | ||
| + | <div class="experience"> | ||
| + | 1% aga gel after PCR under different conditions for A18 (OmpA-Linker from Varsaw). | ||
| + | Expected weight : 525bp. | ||
| + | <center>[[Image:Gel_090812Sylvain.JPG]]</center> | ||
| + | Obviously, something has gone wrong. Next step will be different temperature gradients testing for PCR and/or new matrix. | ||
| + | </div> | ||
| + | |||
| + | <div class="sylvain"> | ||
| + | Transformation | ||
| + | </div> | ||
| + | <div class="experience"> | ||
| + | Classic protocol with DH5a, and pSB2K3 from iGEM DNA plates (1/7C). ON culture on LB + Kan. | ||
| + | |||
| + | To be checked tomorrow ! | ||
| + | </div> | ||
| + | |||
| + | <div class="sylvain"> | ||
| + | ON culture for Minipreps tomorrow | ||
| + | </div> | ||
| + | <div class="experience"> | ||
| + | pSB1A3, pLAC, RBSTetR, OmpA-L, Three ligation clones, ON culture in LB + Abiotics. 37°C with stiring. | ||
| + | </div> | ||
| + | |||
| + | <div class="sylvain"> | ||
| + | ON PCR | ||
| + | </div> | ||
| + | <div class="experience"> | ||
| + | A20 : 49, 51 and 53°C PCR with the plasmid for OmpA-L. Overnigth. 15s elongation time. | ||
| + | </div> | ||
| + | |||
| + | <div class="guillaume"> | ||
| + | All Manip | ||
| + | </div> | ||
| + | <div class="experience"> | ||
| + | Comming soon | ||
</div> | </div> | ||
Latest revision as of 22:37, 16 August 2009
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Lab work
Ligation
D15 (2xterminator E/S) and D16 (pLac E/X) with vector psb2k3
DO : D15=1,19µg/mL D16=1,75µg/mL
| vecteur | insert | H20 | Buffer 10x T4 ligase | T4 ligase | Total | ||
| L5 | 2xterm:pSB2K3(with pLac) | 1µl | 3µl | 13µl | 2µl | 1µl | 20µl |
| control - | o | 1µl | o | 15µl | 2µl | 1µl | 20µl |
1h Home temperature
Transformation
classic protocol with DH5a
Purification
- Purification of A10(ClyA) (PCR at 55° during 30sec) (using 1% agarose during 25 min)
- A10=A10 987pb
- A10 purificated
- Purification of A10 work
PCR Gel verification
1% aga gel after PCR under different conditions for A18 (OmpA-Linker from Varsaw). Expected weight : 525bp.
Obviously, something has gone wrong. Next step will be different temperature gradients testing for PCR and/or new matrix.
Transformation
Classic protocol with DH5a, and pSB2K3 from iGEM DNA plates (1/7C). ON culture on LB + Kan.
To be checked tomorrow !
ON culture for Minipreps tomorrow
pSB1A3, pLAC, RBSTetR, OmpA-L, Three ligation clones, ON culture in LB + Abiotics. 37°C with stiring.
ON PCR
A20 : 49, 51 and 53°C PCR with the plasmid for OmpA-L. Overnigth. 15s elongation time.
All Manip
Comming soon
To do list
| Matricule | TODO |
| Luc | |
| Romain | |
| Charlotte | |
| Stoff | |
| Chris | |
| Lisa | |
| Caroline | |
| Souf | |
| Vicard | |
| Pierre | |
| Sylvain | pSB2K3 transformation ; PCR gels and purifs or new culture for minipreps |
| Guillaume |
