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Re-digestion of plx007, plx008 and plx009

Plasmid 2uL

XhoI 0.5uL

H buffer 2uL

ddH2O 15.5uL

electrophoresis result: invisible

pick colony of plx008, plx009

rescue by precipitation of LacZa

pick lacZa-T positive transformation colony and miniprep

Colony PCR of CD2-T

Preparation of LB medium with Cm

Re-PCR of LacZa and CD2, CD3

primer-F/R 1uL each

Ex-Buffer 5uL

dNTP 5uL

Ex-Taq 0.25uL

ddH2O 36.75uL

60 degrees and 35 cycles with temperature grad

==single digestion of plx007, 008, 009 Plasmid 4uL

XhoI 0.5uL

H buffer 2uL

ddH2O 15.5uL

miniprep of lacZa-T using positive transformation colony and digestion

plasmid 10uL

XhoI/SalI 0.5uL

H buffer 2uL

ddH2O 7uL

total 20uL

digestion for 1 hour and electrophoresis test for the result.

LacZa rescue after digestion

CD2 rescue and ligate with pGEM-T plasmid

single digestion using XhoI of lacZa-T

single digestion of plx007,008,009 using XhoI