Team:HokkaidoU Japan/Notebook/September14

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=Digestion and ligation of pSB1C3, araC Promoter and GFP

Digestion

Electrophoresed digestion product

Used pSB1C3 which was PCRed september 6th. DNA concentration was 2256 ng/uL. So diluted 20 times.

Reagent Amount
10x M buffer 5 uL
DW 34
pSB1C3 1
BSA 5
EcoR I 2
Pst I 3
Total 50 uL
Reagent Amount
10x M buffer 5 uL
DW 27
Promoter 8
BSA 5
EcoR I 3
Spe I 2
Total 50 uL
Reagent Amount
10x M buffer 5 uL
DW 32
GFP 3
BSA 5
Xba I 4
Pst I 1
Total 50 uL

→Incubated at 37C for 120 min
→Added Sample Buffer 10 uL and electrophoresed

  • pSB1C3band wasn't visible

→ Other 2 were gel extracted

  • Diluted that final volume would be 40 uL

Ethanol precipitation

Electrophoresis after ethanol precipitation
  • Added ethanol acetate[3M] 4 uL to 40 uL of previously purified DNA solutuion
  • Added EtOH[100%] 44 uL friezed in dry ice
  • Melted and centrifuged at 15,996rpm, 4C 5 min
  • Discarded supernatant and added EtOH[70%] 100 uL rinsed
  • Centrifuged at 15,996rpm, 4C 5 min
  • Discarded supernatant and dried
  • Diluted in 2 uL of TE each

Anticipated concentrations promoter: 125 ng/2uL GFP: 106 ng/2uL

  • pSB1C3 was cut(EcoR1, Pst1) on September 6th
  • Did ethanol precipitation and electrophoresed to check concentration
  • It was about 20 ng/uL

→Used 2 uL of it

Ligation

Reagent Amount
pSB1C3 2 uL
promoter 2 uL
GFP 2 uL
Mighty Mix 6 uL
T4 ligase 0.4 uL
Total 12.4 uL'
  • 50 uL were transformed by electroporation and remaining 50 uL by heat shock (chemical)