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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