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Team:Paris/17 August 2009
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==Lab work== | ==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 class="charlotte"> | <div class="charlotte"> | ||
Vector Dephosphorylation | Vector Dephosphorylation | ||
Revision as of 15:41, 17 August 2009
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NoteBook
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Brain work
edit please ^^
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)
To do list
edit please ^^
