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Team:Paris/17 August 2009
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==NoteBook== | ==NoteBook== | ||
| - | + | {{Paris2009_Calendar}} | |
| - | { | + | {{Paris2009_Calendar_Link|16_August_2009|18_August_2009}} |
| - | + | <center> '''August 17th''' </center> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
<html> | <html> | ||
| Line 19: | Line 14: | ||
</html> | </html> | ||
| + | ==Lab work== | ||
| - | == | + | <div class="romain"> |
| + | Competent Bacteria | ||
| + | </div> | ||
| + | <div class="experience"> | ||
| + | *Competent bacteria | ||
| + | **1/50 ON culture of New DH5α | ||
| + | **OD<sub>600</sub>=0,6 | ||
| + | *Transformation with puc19 (10pg/µl) | ||
| + | **250µl of competent DH5α | ||
| + | **1µl puc19 | ||
| + | **Leave 20 min in ice | ||
| + | **45s at 42°C | ||
| + | **Leave 2 min in ice | ||
| + | **Add 1ml of hot LB (42°C) | ||
| + | **1h under agitation | ||
| + | **Put on plate (LB agar + Amp) | ||
| - | |||
| - | |||
| - | |||
| - | |||
</div> | </div> | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
<div class="charlotte"> | <div class="charlotte"> | ||
Vector Dephosphorylation | Vector Dephosphorylation | ||
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<div class="experience"> | <div class="experience"> | ||
| - | |||
pSB1A3 digested by '''EcoRI/PstI''', '''XbaI/PstI''' and '''EcoRI/SpeI''' have to be dephosphorylated before the ligation process. | pSB1A3 digested by '''EcoRI/PstI''', '''XbaI/PstI''' and '''EcoRI/SpeI''' have to be dephosphorylated before the ligation process. | ||
| - | Mix : | + | |
| + | ''Mix'' : | ||
| + | |||
Vector : 30uL | Vector : 30uL | ||
| Line 51: | Line 53: | ||
Buffer : 3,5 uL | Buffer : 3,5 uL | ||
| + | |||
| + | ''Protocol'' | ||
Put the solution at 37°C for 30 min | Put the solution at 37°C for 30 min | ||
| Line 61: | Line 65: | ||
</div> | </div> | ||
| + | |||
<div class="charlotte"> | <div class="charlotte"> | ||
| - | + | Ligation | |
</div> | </div> | ||
<div class="experience"> | <div class="experience"> | ||
| + | |||
| + | |||
| + | [https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D13 D13] (X/P): 0,94 ug/mL | ||
| + | |||
| + | [https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D14 D14] (X/P): 1,2 ug/ml | ||
| + | |||
| + | [https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#P16 pSB1A3] (X/P): 2,1 ug/ml | ||
| + | |||
| + | |||
| + | Ligation (2hrs at RT) of pSB1A3 w/ D13 (ompA signal) or D14 (TolRII) | ||
| + | |||
| + | |||
| + | ''Mix : total volume = 10 uL'' | ||
| + | |||
| + | vector (pSB1A3) : 2 uL | ||
| + | |||
| + | insert (A11 or A13) : 4uL | ||
| + | |||
| + | 10X T4DNA Ligase buffer : 1 uL | ||
| + | |||
| + | T4 DNA Ligase : 0,5 uL | ||
| + | |||
| + | H2O: 2,5 uL | ||
| + | |||
| - | + | ''Negatif control '': same mix as the previous one but without the insert. In these conditions the amount of water was increased until 6,5 uL. | |
</div> | </div> | ||
| Line 74: | Line 103: | ||
<div class="charlotte"> | <div class="charlotte"> | ||
| - | + | Transformation | |
</div> | </div> | ||
<div class="experience"> | <div class="experience"> | ||
| - | Ligation of | + | '''''Transformation of (using the "heat choc" protocol)''''' : |
| + | |||
| + | - BBa_B0034 (''AmpR'') | ||
| + | |||
| + | - BBa_E0030(''KanR'') | ||
| + | |||
| + | - pSB3C5 (''CmR'') | ||
| + | |||
| + | - pINTE3 (Col E3, ''AmpR'') | ||
| + | |||
| + | - pTA (pTet-Col A, ''AmpR'') | ||
| + | |||
| + | - pINTg3p (g3p Term, ''AmpR'') | ||
| + | |||
| + | - pINK2(TolRII, ''AmpR'') | ||
| + | |||
| + | - Ligation 1 ( set up on Friday 14th of august w/ [https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D11 D11 (RBS-Tet digested by Xba/Pst)] and vecteur [https://2009.igem.org/Team:Paris/Freezer_Digestion_Products#D12 D12 (pSB2K3 w/ pLac digested by Spe/Pst)] (''KanR'') | ||
| + | |||
| + | - previous ligation (w/ D13 and D14) (''AmpR'') | ||
</div> | </div> | ||
| - | == | + | <div class="vicard"> |
| + | Transformation Check/PCR on colonies | ||
| + | </div> | ||
| + | <div class="experience"> | ||
| + | <strong>Transformation check:</strong> | ||
| + | |||
| + | The x10mix ligation after centrifugation and put at 16°C overnight had work. | ||
| + | |||
| + | using less incubation time at RT seems to work better. | ||
| + | |||
| + | using less insert seems to work better | ||
| + | |||
| + | <strong>PCR on colonies:</strong> | ||
| + | |||
| + | 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 3''' | ||
| + | <center>[[Image:3e_colobis.JPG]]</center> | ||
| + | |||
| + | |||
| + | </div> | ||
| + | |||
| + | |||
| - | + | {{Paris2009_Calendar_Link|16_August_2009|18_August_2009}} | |
Latest revision as of 09:38, 7 September 2009
NoteBook
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Lab work
Competent Bacteria
- Competent bacteria
- 1/50 ON culture of New DH5α
- OD600=0,6
- Transformation with puc19 (10pg/µl)
- 250µl of competent DH5α
- 1µl puc19
- Leave 20 min in ice
- 45s at 42°C
- Leave 2 min in ice
- Add 1ml of hot LB (42°C)
- 1h under agitation
- Put on plate (LB agar + Amp)
Vector Dephosphorylation
pSB1A3 digested by EcoRI/PstI, XbaI/PstI and EcoRI/SpeI have to be dephosphorylated before the ligation process.
Mix :
Vector : 30uL
Antarctic Phosphatase : 2uL
Buffer : 3,5 uL
Protocol
Put the solution at 37°C for 30 min
Add 2 uL of enzyme
37°C for 30 min
65°C for 20 min for heat inactivation
Ligation
D13 (X/P): 0,94 ug/mL
D14 (X/P): 1,2 ug/ml
pSB1A3 (X/P): 2,1 ug/ml
Ligation (2hrs at RT) of pSB1A3 w/ D13 (ompA signal) or D14 (TolRII)
Mix : total volume = 10 uL
vector (pSB1A3) : 2 uL
insert (A11 or A13) : 4uL
10X T4DNA Ligase buffer : 1 uL
T4 DNA Ligase : 0,5 uL
H2O: 2,5 uL
Negatif control : same mix as the previous one but without the insert. In these conditions the amount of water was increased until 6,5 uL.
Transformation
Transformation of (using the "heat choc" protocol) :
- BBa_B0034 (AmpR)
- BBa_E0030(KanR)
- pSB3C5 (CmR)
- pINTE3 (Col E3, AmpR)
- pTA (pTet-Col A, AmpR)
- pINTg3p (g3p Term, AmpR)
- pINK2(TolRII, AmpR)
- Ligation 1 ( set up on Friday 14th of august w/ D11 (RBS-Tet digested by Xba/Pst) and vecteur D12 (pSB2K3 w/ pLac digested by Spe/Pst) (KanR)
- previous ligation (w/ D13 and D14) (AmpR)
Transformation Check/PCR on colonies
Transformation check:
The x10mix ligation after centrifugation and put at 16°C overnight had work.
using less incubation time at RT seems to work better.
using less insert seems to work better
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 3
