Team:Paris/17 August 2009
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Cha.olivier (Talk | contribs) (→Lab work) |
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Put the solution at 37°C for 30 min | Put the solution at 37°C for 30 min |
Revision as of 13:16, 17 August 2009
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Brain work
edit please ^^
Lab work
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 ^^