Ligation of SulA, Rec, SD1-3, SS1-2 with GFP-pLX007
Precipitate for retraction
SulA, Rec, SD1-3, SS1-2, GFP-pLX007 (^^ with XbaI & XhoI)
Ligate with GFP-pLX007
SulA, Rec, SD1-3, SS1-2
Transformed in the evening
Colony PCR test of Prec, SD2-3, SS1-3-(SDY-EGFP-pLX007)
Number of colonies grew up:
No. ---------- Prec 0 SD2 0 SD3 1 SS1 >50 SS2 2 SS3 14
Only SS1 is right. 4 colonies are select for incubation.
Colony PCR test of T1T2-(Tet F-lacZ-pLX010)
22 colonies are selected for incubation.
PCR and double digestion of RD1
PCR of RD1
Retract through electropheresis.
Double digestion of RD1
10uL 10*H buffer 2uL SpeI 2uL XhoI 15uL RD1 71uL ddH2O ------------------- 100uL total