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Team:Paris/18 August 2009
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Revision as of 14:07, 18 August 2009
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NoteBook
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Brain work
edit please ^^
Lab work
PCR on colony w/ Soufifi
The ligation pLAC/RBS-TetR worked --> no clone on the negatif control and some clone on the "good" plates.
PCR on colony is required to check the validity of our clones.
Mix : total volume = 25uL
Super Mix 2X: 12,5 uL
VF2 (10uM): 0,5 uL
VR (10 uM): 0,5 uL
H20 : 6,5 uL
diluted bacteria : 5 uL
Tm = 55°C
Elongation time = 1 min
size expected :
- pLac/RBS-TetR : +/- 700 bp
- VF2 and VR plasmid amplification : +/- 150 bp
- final size : +/- 850 bp
Digestion
The ligation that I have set up yesterday (pSB1A3 w/ D13 or D14 ) didn't work so I decided to redigest PCR products (A11 (RBS-ompA signal = D13) and A13 (TolRII = D14)).
Mix : total volume = 30 uL
For A11 (RBS-OmpA signal):
DNA = 15 uL
10X buffer: 3 uL
SpeI: 1 uL
PstI: 1 uL
BSA: 0,5 uL
H2O: 9,5 uL
For A13:
DNA = 10 uL
10X buffer: 3 uL
XbaI: 1 uL
PstI: 1 uL
BSA: 0,5 uL
H2O: 14,5 uL
Other digestions were done :
- The double terminator (BBa_0034) by EcoRI/XbaI and SpeI/PstI (same mix as the one for A13)
- The pTet (BBa_....) by SpeI/PstI and EcoRI/PstI
Gel purification w/ Bobode
Promega kit, 3 uL loadded
PCR : 70,2°C 9s elongation
To do list
edit please ^^
