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Team:Paris/11 August 2009
From 2009.igem.org
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| - | <center>[[ | + | <center>[[Image:Digestion_1.JPG]]</center> (15min migration) |
| - | <center>[[ | + | <center>[[Image:Digestion_2.JPG]]</center> (35min migration) |
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Revision as of 17:03, 11 August 2009
Contents |
NoteBook
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Brain work
edit please ^^
Lab work
Digestion
A11 (OmpAsignal) and A13 (TolRII)
| DNA | Buffer 10X | H20 | BSA | XbaI | PstI | Total |
| 20µl | 3µl | 4,5µl | 0,5µl | 1µl | 1µl | 30µl |
2h 37°C
20min 65°C (stop enzymes)
purification
Ligation
D13 (OmpAsignal X/P) and D14 (TolRII X/P) and vecteur pSB1A3 digested with XbaI and PstI
DO : D13=0,94 D14=1,29 pSB1A3=0,74
| vecteur | insert | H20 | Buffer 10x T4 ligase | T4 ligase | Total | ||
| L3 | TolRII:pSB1A3 | 2µl | 3µl | 12µl | 2µl | 1µl | 20µl |
| L4 | OmpAsignal:pSB1A3 | 2µl | 3µl | 12µl | 2µl | 1µl | 20µl |
| control - | o | 2µl | o | 15µl | 2µl | 1µl | 20µl |
1h Home temperature
Transformation
classic protocol with DH5a
Ligation
D11 (RBS-Tet digested by Xba/Pst) and vecteur D12 (pSB2K3 w/ pLac digested by Spe/Pst)
DO : D11= D12=
| vecteur | insert | H20 | Buffer 10x T4 ligase | T4 ligase | Total | ||
| L1 | D11 | µl | µl | µl | µl | µl | µl |
| control - | o | µl | o | µl | µl | µl | µl |
PCR
- For A10(ClyA) because we have 2 bands in the last gel migration of A3(ClyA) purification (06/08/09)
- without DMSO
- with [010] and [031]
- 1:98°C 1min
- 2:98°C 10s
- 3:55°C, 60°C, and 65°C 30s
- 4:72°C 30s
- 5:72°C 10min
- 6: 4°C ~
- Gel-->[Ladder 1kb/A10 at 55°C/A10 at 60°C/A10 at 65°C]
- A10 at 55°C work the best we have the biggest and brightest band with this temperature.
Digestion
- Digestion of P4 double terminator with EcoRI/SpeI (D15)in order to ligate with P21 (double terminator)
- 20µl of P21
- 2µl of EcoR1 and 2µl of SpeI
- 0,5µl BSA 100X
- 5µl buffer 2 x10
- 20,5µl H20
- Vfinal=50µl
- 2h at 37°C
- Digestion of P21 (pLac in pSB2K3) with EcoRI/XbaI (D16)in order to ligate with P4 (double terminator)
- 20µl of P21
- 2µl of EcoR1 and 2µl of Xba1
- 0,5µl BSA 100X
- 5µl buffer 2 x10
- 20,5µl H20
- Vfinal=50µl
- 2h at 37°C
- Gel-->[Ladder 100pb/D15/Ladder 1kb/D16]
To do list
| Matricule | TODO |
| Luc | |
| Romain | |
| Charlotte | |
| Stoff | |
| Chris | |
| Lisa | |
| Caroline | |
| Souf | |
| Vicard | |
| Pierre | |
| Sylvain | |
| Guillaume |
