Team:HokkaidoU Japan/Notebook/August30

From 2010.igem.org

Notebook

Digestion of pUC119 by EcoR I, Pst I

－ EcoR I Pst I E, P Old EcoR I
DNA solution 1 uL 1 uL 1 uL 1 uL 1 uL
DW 17 uL 14 uL 14 uL 13 uL 14 uL
10x M buffer 2 uL 2 uL 2 uL 2 uL 2 uL
0.1% BSA － 2 uL 2 uL 2 uL 2 uL
EcoR I － 1 uL － 1 uL 1 uL
Pst I － － 1 uL 1 uL －
Total 20 uL 20 uL 20 uL 20 uL 20 uL

→Incubated at 37C for 60 min →Electrophoresed 2 uL for confirmation

  • There were no bands, forgot to add DNA
  • Reused the remaining 18 uL of digestion solution by adding 1 uL of ADW and 1uL of DNA

→Incubated at 37C for 60 min
→Electrophoresed 2 uL of each solution（+ 0.4 uL 6x SB）

Electrophoresis

Electrophoresis of digestion products
Lane DNA
2 λ/HindIII,　EcoR I（4 uL used）
3 Undigested
4 EcoR I
5 Pst I
6 EcoR I + Pst I
7 EcoR I (used old enzyme to check it's activity)
  • In lane 3 monomers, dimers and trimers of plasmid were visible ．
  • From lanes 4 through 7 it's visible that DNA digestion wasn't satisfactory
    • Because plasmid became linear it's was slower than super-coiled one's

Digestion of parts PCRed using digestion visualization primers

　 RBS double terminator GFP
DNA 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL
DW 17 uL 14 uL 14 uL 13 uL 17 uL 14 uL 14 uL 13 uL 17 uL 14 uL 14 uL 13 uL
10x M buffer 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL
0.1% BSA － 2 uL 2 uL 2 uL － 2 uL 2 uL 2 uL － 2 uL 2 uL 2 uL
EcoR I － 1 uL － 1 uL － 1 uL － 1 uL － 1 uL － 1 uL
Pst I － － 1 uL 1 uL － － 1 uL 1 uL － － 1 uL 1 uL
Total 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL
Electrophoresis of digestion products

→Incubated at 37C for 60 min

Electrophoresis

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