IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-20

double digesting E0040 and I7100 for ligation

 * Digesting E0040 and I7100 with EcoR1/SpeI.
 * Digestion system contains (totally 19uL): 2uL 10*H, 0.5uL EcoRI, 0.5uL PstI, 6uL plasmid, 10uL ddH2O.
 * 37℃ culutre for 12 hours.

electrophoresis result

 * from left to right:
 * 1   I7100 plasmid
 * 2-5 I7100 and EcoR1 and Pst1
 * 6   E0040 plasmid
 * 7-10 E0040 and EcoR1 and Pst1
 * 11  marker DL200 plus

Gel Purification

 * extract pSB3k3(I7100) vector and e0040 fragments from gel

electrophoresis test the products of gel extraction

 * Digestion system contains:2uL plasmid,2uL Loading Buffer,2uL Dye.
 * from left to right:
 * 1) EGFP(E0040)
 * 2) pSB3k3(I7100)
 * 3) Marker DL200 plus

ligation

 * ligate the pSB3k3(I7100) and EGFP(E0040) with T4 ligase

single digestion test

 * Digesting E0040 and r0040 with EcoR1.
 * Digestion system contains (totally 20uL): 2uL 10*H, 0.5uL EcoRI, 4uL plasmid, 13.5uL dH2O.
 * 37℃ culutre for 3 hours.

electrophoresis result

 * from left to right:
 * 1) 1-3 e0040 and EcoR1
 * 2) 4-5 r0040 and EcoR1
 * 3) 6 Marker


 * ! there are abnormal bands in the e0040 samples.

another single digestion test

 * digest I7100 with Xba1
 * digest e0040(another sample) with EcoR1
 * Digestion system contains (totally 20uL):
 * 1) I7100: 2uL 10*M, 0.5uL XbaI, 4uL plasmid, 13.5uL dH2O.
 * 2) E0040: 2uL 10*H, 0.5uL EcoRI, 4uL plasmid, 13.5uL dH2O.

electrophoresis result

 * From left to right:
 * 1) 1 I7100 plasmid
 * 2) 2-4 I7100 and Xba1
 * 3) 5 e0040 plasmid
 * 4) 6-8 e0040 and EcoR1
 * 5) 9 marker DL2000 plus
 * 6) 10 r0040 and EcoR1 (products of former digestion test)