IGEM:Peking/2007/Switch-Notebook/2007-8-30
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Cultivate E.coli
bacteria with sulA, RS2, RD3-plx012
bacteria with pSB3K3, pSB1A3 and pcc056
PCR 434OAR321 and 434OR21
PCR system:
Ex-Taq buffer 5uL
dNTP 5uL
template 1uL
primer F/R 1uL each
Ex-Taq 0.25uL
ddH2O 36.75uL
total system is 50uL
result: we get the fragments!
colony PCR of T1TE-T1T2-pcc010 and CD2-plx012
enzymatic test of c0056 and pSB3K3
using EcoRI and PstI in H buffer
miniprep of lacIq-T
digestion test with XhoI/SalI in H buffer
Miniprep of MCF 1,2,3
digestion test with BamHI/XhoI in K buffer
ligation of lacIq with pcc009
4 degrees overnight
434OR321/21 ligates with T vector
transformation to E.coli after ligation
I13521 digestion
using EcoRI and PstI in H buffer, overnight