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Team:Paris/15 August 2009
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==NoteBook== | ==NoteBook== | ||
| - | { | + | {{Paris2009_Calendar}} |
| - | + | {{Paris2009_Calendar_Link|14_August_2009|16_August_2009}} | |
| - | + | <center> '''August 15th''' </center> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
<html> | <html> | ||
| Line 19: | Line 15: | ||
</html> | </html> | ||
| - | + | ==Lab work== | |
| - | == | + | <div class="guillaume"> |
| - | + | Transformation | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | < | + | |
</div> | </div> | ||
| - | <div | + | <div class="experience"> |
| + | *For overnight (at 16°C) ligation solution of g3p in pSB1A3 | ||
| + | *For overnight (at 16°C) ligation solution of ClyA in pSB1A3 | ||
</div> | </div> | ||
| - | <div | + | <div class="guillaume"> |
| - | + | Miniprep | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
</div> | </div> | ||
| - | <div | + | <div class="experience"> |
| + | *ptet | ||
| + | *pBad/araC | ||
| + | *Double terminater | ||
| + | </div> | ||
| + | <div class="guillaume"> | ||
| + | Glycerol Stock | ||
| + | </div> | ||
| + | <div class="experience"> | ||
| + | *New strains | ||
| + | **[S35] AA93 ΔfecA | ||
| + | **[S36] fecIR mutant | ||
| + | **[S37] fecIR wt | ||
| + | **[S38] fecA | ||
| + | </div> | ||
| + | <div class="guillaume"> | ||
| + | Yesterday transformations results | ||
| + | </div> | ||
| + | <div class="experience"> | ||
| + | *tatABC | ||
| + | **No bacteria | ||
| + | *pFecA | ||
| + | **Negative control = 0 bacteria | ||
| + | **1:1 raport = 43 bacteria | ||
| + | **1:7 raport = 2 bacteria | ||
| + | **1:7 concentrated = Too much | ||
| + | *g3p | ||
| + | **Negative control = 0 bacteria | ||
| + | **1:1 raport = 1 bacteria | ||
| + | **1:7 raport = 0 bacteria | ||
| + | **1:7 concentrated = 16 bacteria | ||
| + | </div> | ||
| + | <div class="guillaume"> | ||
| + | PCR screening protocol | ||
| + | </div> | ||
| + | <div class="experience"> | ||
| + | *Program named COLONIES, use with VF2 and VR oligos | ||
| + | **Lid 105°C | ||
| + | **1: 99°C 2min | ||
| + | **2: Sound | ||
| + | **3: Pause until press "Enter" button | ||
| + | **4: 95°C 5min | ||
| + | **5: 95°C 30sec | ||
| + | **6: 55°C 30sec | ||
| + | **7: 72°C [45sec] (1kb/bp, caution the universal oligo add 156bp) | ||
| + | **8: Goto 5 29x | ||
| + | **9:72°C 7min | ||
| + | **10: Hold at 4°C | ||
| + | *Preparation of the ON culture (for Glycerol stock and Miniprep of the positives clones) | ||
| + | **7ml of LB+ Amp (because we use pSB1A3 as vector) | ||
| + | **Bacterial solution | ||
| + | **100uL of H2O and few bacteria in a PCR tube | ||
| + | **Put the cone in LB solution for ON culture | ||
| + | **Put the PR tube in PCR and run the fist part of the program | ||
| + | *PCR mix | ||
| + | **Master Mix (2x) : 12,5ul | ||
| + | **Oligo VF2 (10uM) : 0,5ul | ||
| + | **Oligo VR (10uM) : 0,5ul | ||
| + | **H2O : 6,5ul | ||
| + | **Bacteria hoted at 99°C by PCR program : 5ul | ||
| + | *Put PCR tube withe the PCR mix in the PCR and run the second part of the program (just push the "Enter" button) | ||
| + | </div> | ||
| + | <div class="guillaume"> | ||
| + | PCR screening | ||
| + | </div> | ||
| + | <div class="experience"> | ||
| + | *For 6 clones of pFecA ligation in pSB1A3 | ||
| + | *Negative control without bacteria | ||
| + | *"Positive" control with pSB1A3 wich have the mRFP (1050bp) | ||
| + | </div> | ||
| + | <div class="guillaume"> | ||
| + | Gel migration of the PCR screening | ||
| + | </div> | ||
| + | <div class="experience"> | ||
| + | *All the clone have se same profil (more than 300bp and under 400bp) and it seem to be good...Confirmation tomorow by enzymes digestion. | ||
| + | *Negative control is...negative | ||
| + | *Positive control is...positif but have a second band. (Must be confirmed) | ||
| + | <center>[[Image:150809 Colony PCR pFecA.png]]</center> | ||
</div> | </div> | ||
| - | |||
| - | |||
| - | |||
| - | + | {{Paris2009_Calendar_Link|14_August_2009|16_August_2009}} | |
Latest revision as of 15:04, 24 August 2009
NoteBook
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Lab work
Transformation
- For overnight (at 16°C) ligation solution of g3p in pSB1A3
- For overnight (at 16°C) ligation solution of ClyA in pSB1A3
Miniprep
- ptet
- pBad/araC
- Double terminater
Glycerol Stock
- New strains
- [S35] AA93 ΔfecA
- [S36] fecIR mutant
- [S37] fecIR wt
- [S38] fecA
Yesterday transformations results
- tatABC
- No bacteria
- pFecA
- Negative control = 0 bacteria
- 1:1 raport = 43 bacteria
- 1:7 raport = 2 bacteria
- 1:7 concentrated = Too much
- g3p
- Negative control = 0 bacteria
- 1:1 raport = 1 bacteria
- 1:7 raport = 0 bacteria
- 1:7 concentrated = 16 bacteria
PCR screening protocol
- Program named COLONIES, use with VF2 and VR oligos
- Lid 105°C
- 1: 99°C 2min
- 2: Sound
- 3: Pause until press "Enter" button
- 4: 95°C 5min
- 5: 95°C 30sec
- 6: 55°C 30sec
- 7: 72°C [45sec] (1kb/bp, caution the universal oligo add 156bp)
- 8: Goto 5 29x
- 9:72°C 7min
- 10: Hold at 4°C
- Preparation of the ON culture (for Glycerol stock and Miniprep of the positives clones)
- 7ml of LB+ Amp (because we use pSB1A3 as vector)
- Bacterial solution
- 100uL of H2O and few bacteria in a PCR tube
- Put the cone in LB solution for ON culture
- Put the PR tube in PCR and run the fist part of the program
- PCR mix
- Master Mix (2x) : 12,5ul
- Oligo VF2 (10uM) : 0,5ul
- Oligo VR (10uM) : 0,5ul
- H2O : 6,5ul
- Bacteria hoted at 99°C by PCR program : 5ul
- Put PCR tube withe the PCR mix in the PCR and run the second part of the program (just push the "Enter" button)
PCR screening
- For 6 clones of pFecA ligation in pSB1A3
- Negative control without bacteria
- "Positive" control with pSB1A3 wich have the mRFP (1050bp)
Gel migration of the PCR screening
- All the clone have se same profil (more than 300bp and under 400bp) and it seem to be good...Confirmation tomorow by enzymes digestion.
- Negative control is...negative
- Positive control is...positif but have a second band. (Must be confirmed)
