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Team:Paris/18 August 2009
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==NoteBook== | ==NoteBook== | ||
| - | { | + | {{Paris2009_Calendar}} |
| - | + | {{Paris2009_Calendar_Link|17_August_2009|19_August_2009}} | |
| - | + | <center> '''August 18th''' </center> | |
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''Mix : total volume = 25uL'' | ''Mix : total volume = 25uL'' | ||
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Super Mix 2X: 12,5 uL | Super Mix 2X: 12,5 uL | ||
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Elongation time = 1 min | Elongation time = 1 min | ||
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| + | '''size expected : ''' | ||
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| + | - ''pLac/RBS-TetR '': +/- 700 bp | ||
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| + | - ''VF2 and VR plasmid amplification'' : +/- 150 bp | ||
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| + | - ''final size'' : +/- '''850 bp''' | ||
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| + | <center>[[Image:PCRcoloy pLac RBS-TetR.PNG]]</center> | ||
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</div> | </div> | ||
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<div class="charlotte"> | <div class="charlotte"> | ||
| + | Gel purification w/ Bobode | ||
| + | </div> | ||
| + | <div class="experience"> | ||
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| + | Promega kit, 3 uL loadded | ||
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| + | <center>[[Image:Gel purif.PNG]]</center> | ||
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| + | </div> | ||
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| + | <div class="vicard"> | ||
Gel purification | Gel purification | ||
</div> | </div> | ||
<div class="experience"> | <div class="experience"> | ||
| - | + | <strong>PCR on colonies:</strong> | |
| + | using for 1 tube: | ||
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| + | Master Mix 2X : 12,5µL | ||
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| + | Oligo VF2 (10µM): 0,5µL | ||
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| + | Oligo VR (10µM): 0,5µL | ||
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| + | bacteria in H20 (see PCR on colonies protocol): 5µL | ||
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| + | H20 : 6,5µL | ||
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| + | '''PCR on colonies Check OK for colonie 7, 8, 9 and 10''' | ||
| + | <center>[[Image:7810colo.JPG]]</center> | ||
| + | |||
| + | Control+ (with PSB1A3) | ||
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| + | Colonie 7, 8 and 10 OK. | ||
</div> | </div> | ||
| - | == | + | <div class="vicard"> |
| + | Stock Glycerol | ||
| + | </div> | ||
| + | <div class="experience"> | ||
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| + | stock glycerol for colonie 3 ClyA in PSB1A3 using: | ||
| + | -1/3 glycerol (333µL) + 2/3 bacteria (667µL) | ||
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| + | </div> | ||
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| + | <div class="vicard"> | ||
| + | Miniprep for colonie 3, 7, 8 and 10 | ||
| + | </div> | ||
| + | <div class="experience"> | ||
| + | |||
| + | Miniprep for colonie 3, 7, 8 and 10 | ||
| + | |||
| + | </div> | ||
| - | + | {{Paris2009_Calendar_Link|17_August_2009|19_August_2009}} | |
Latest revision as of 10:46, 7 September 2009
NoteBook
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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
Gel purification
PCR on colonies:
using for 1 tube:
Master Mix 2X : 12,5µL
Oligo VF2 (10µM): 0,5µL
Oligo VR (10µM): 0,5µL
bacteria in H20 (see PCR on colonies protocol): 5µL
H20 : 6,5µL
PCR on colonies Check OK for colonie 7, 8, 9 and 10
Control+ (with PSB1A3)
Colonie 7, 8 and 10 OK.
Stock Glycerol
stock glycerol for colonie 3 ClyA in PSB1A3 using: -1/3 glycerol (333µL) + 2/3 bacteria (667µL)
Miniprep for colonie 3, 7, 8 and 10
Miniprep for colonie 3, 7, 8 and 10
