CD2-T and T-1 send to sequencing
T1TE and T1T2
T1TE digestion by ClaI/SalI and T1T2 digestion by NotI/XhoI, then rescued.
NdeI and XhoI digestion using H buffer, send to sequencing.
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