Team:Paris/17 August 2009
From 2009.igem.org
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Brain work
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Lab work
Vector Dephosphorylation
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
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 BBa_B0034 (AmpR), BBa_E0030(KanR), pSB3C5 (CmR), pINTE3 (Col E3, AmpR), pTA (pTet-Col A, AmpR), pINTg3p (g3p Term, AmpR), pINK2(TolRII, AmpR) and previous ligation using the "heat choc" protocol
To do list
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