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CD2-T and T-1 send to sequencing

T1TE and T1T2

T1TE digestion by ClaI/SalI and T1T2 digestion by NotI/XhoI, then rescued.

LacZa-plx009 miniprep

NdeI and XhoI digestion using H buffer, send to sequencing.

sequencing result

plx008 is right

plx009-5 turned out to be plx003!!!

So actually we have already constructed lacZa-Plx003!

But there is still some problems with enzymatic digestion results.

We will change our construction plan due to this result!

plx006 digestion using XhoI and H buffer

result: plx006 can not be cut by XhoI wihout any known reason.

CD2, RD3, RS3, CD3 PCR and rescue

using relevant primers to PCR and then rescue by gel.

==plx007/8 transform JM110/DH5a

T1TE and T1T2 ligate with pcc010

4 degrees in fridge overnight with a concentration ratio of 4:1 between fragment and vector