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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:

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