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Team:Paris/12 August 2009
From 2009.igem.org
NoteBook
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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 |
