Team:Paris/16 August 2009
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==NoteBook== | ==NoteBook== | ||
- | { | + | {{Paris2009_Calendar}} |
- | + | {{Paris2009_Calendar_Link|15_August_2009|17_August_2009}} | |
- | + | <center> '''August 16th''' </center> | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
<html> | <html> | ||
</div> | </div> | ||
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<div id="paris_content"> | <div id="paris_content"> | ||
</html> | </html> | ||
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+ | ==Lab work== | ||
+ | <div class="guillaume"> | ||
+ | Yesterday transformations results | ||
+ | </div> | ||
+ | <div class="experience"> | ||
+ | *g3p ON ligation at 16°C | ||
+ | **Negative control concentrated = 0 bacteria | ||
+ | **1:1 concentrated = 1 bacteria | ||
+ | **1:7 concentrated = 1 bacteria | ||
+ | *ClyA ON ligation at 16°C | ||
+ | **Negative control concentrated = 0 bacteria | ||
+ | **1:1 rapport = 0 bacteria | ||
+ | **1:7 rapport = 0 bacteria | ||
+ | **1:1 concentrated = 10 bacteria | ||
+ | **1:7 concentrated = 0 bacteria | ||
+ | </div> | ||
+ | <div class="guillaume"> | ||
+ | Miniprep | ||
+ | </div> | ||
+ | <div class="experience"> | ||
+ | *For the 6 positives clone of pFecA | ||
+ | </div> | ||
+ | <div class="guillaume"> | ||
+ | Glycerol Stock | ||
+ | </div> | ||
+ | <div class="experience"> | ||
+ | *For clone n°1 and n°2 | ||
+ | **333ul of Glycerol 60% | ||
+ | **666ul of ON culture | ||
+ | **Mix and store at -80°C | ||
+ | </div> | ||
+ | <div class="guillaume"> | ||
+ | Digestion | ||
+ | </div> | ||
+ | <div class="experience"> | ||
+ | *Digestion for the 6 clones of pFecA ligation, by XbaI and PstI | ||
+ | *Mix | ||
+ | **DNA (Miniprep) : 5ul | ||
+ | **XbaI : 1ul (I think it's too much...but I think to this after doing it) | ||
+ | **PstI : 1ul | ||
+ | **BSA 100x : 0,2ul | ||
+ | **Buffer NEB2 10x : 2ul | ||
+ | **H2O : 9ul | ||
+ | *2h at 37°C | ||
+ | </div> | ||
+ | <div class="guillaume"> | ||
+ | Gel migration | ||
+ | </div> | ||
+ | <div class="experience"> | ||
+ | *1,5% agarose gel | ||
+ | *5ul drop | ||
+ | *Good profil, expected band at 130bp. Maybe not enough digest bu we clearly see the 130bp band corresponding to the insert (pFecA). The 2000bp band correspond to pSB1A3 | ||
<center>[[Image:Wiki 160809 pFecA digest after ligation.png]]</center> | <center>[[Image:Wiki 160809 pFecA digest after ligation.png]]</center> | ||
+ | </div> | ||
+ | <div class="guillaume"> | ||
+ | ON culture | ||
+ | </div> | ||
+ | <div class="experience"> | ||
+ | *DH5α in order to done more competent bacteria (the stock is shrinking) | ||
+ | *4 g3p clones (7ml + 7ul Amp) | ||
+ | </div> | ||
+ | <div class="guillaume"> | ||
+ | PCR screening | ||
+ | </div> | ||
+ | <div class="experience"> | ||
+ | *Same protocol as yesterday | ||
+ | *Screening of 4 clones (maybe I invert the n°2 and the n°4) | ||
+ | </div> | ||
+ | <div class="guillaume"> | ||
+ | Gel migration | ||
+ | </div> | ||
+ | <div class="experience"> | ||
+ | *g3p is around 1450bp... Second confirmation tomorrow but it seems very good! | ||
+ | <center>[[Image:Wiki 160809 PCRcolonies g3p.png]]</center> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | {{Paris2009_Calendar_Link|15_August_2009|17_August_2009}} |
Latest revision as of 15:05, 24 August 2009
NoteBook
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Lab work
Yesterday transformations results
- g3p ON ligation at 16°C
- Negative control concentrated = 0 bacteria
- 1:1 concentrated = 1 bacteria
- 1:7 concentrated = 1 bacteria
- ClyA ON ligation at 16°C
- Negative control concentrated = 0 bacteria
- 1:1 rapport = 0 bacteria
- 1:7 rapport = 0 bacteria
- 1:1 concentrated = 10 bacteria
- 1:7 concentrated = 0 bacteria
Miniprep
- For the 6 positives clone of pFecA
Glycerol Stock
- For clone n°1 and n°2
- 333ul of Glycerol 60%
- 666ul of ON culture
- Mix and store at -80°C
Digestion
- Digestion for the 6 clones of pFecA ligation, by XbaI and PstI
- Mix
- DNA (Miniprep) : 5ul
- XbaI : 1ul (I think it's too much...but I think to this after doing it)
- PstI : 1ul
- BSA 100x : 0,2ul
- Buffer NEB2 10x : 2ul
- H2O : 9ul
- 2h at 37°C
Gel migration
- 1,5% agarose gel
- 5ul drop
- Good profil, expected band at 130bp. Maybe not enough digest bu we clearly see the 130bp band corresponding to the insert (pFecA). The 2000bp band correspond to pSB1A3
ON culture
- DH5α in order to done more competent bacteria (the stock is shrinking)
- 4 g3p clones (7ml + 7ul Amp)
PCR screening
- Same protocol as yesterday
- Screening of 4 clones (maybe I invert the n°2 and the n°4)
Gel migration