Team:HokkaidoU Japan/Notebook/August16

From 2010.igem.org

Notebook

Restriction Enzyme Activity Check

Restriction Enzyme Digestion

EcoR I, Xba I, Pst I

Reagent Amount
pUC119 1 uL
DW 7 uL
10x M Buffer 1 uL
Restriction Enzyme 1 uL
Total 10 uL
  • Made solution for each EcoR I, Xba I, Pst I
    • incubated at 37C for 60 min

Spe I

Reagent Amount
1-1A 1 uL
DW 7 uL
10x M Buffer 1 uL
Restriction Enzyme 1 uL
Total 10 uL
  • pUC119 doesn't have Spe I restriction sites, so we used mini preped plasmid with biobrick insert
    • incubated at 37C for 60 min

Control

  • Added 1 uL of 6x Sample Buffer to 1 uL of pUC119
  • Also added 1 uL of 6x Sample Buffer to 1 uL of 1-1A
    • Incubated at 37C for 60 min

Electrophoresis

Electrophoresis of digested DNA

Added 2 uL of 6x Sample Buffer to digested solution and performed electrophoresis

Lane DNA
1 λ/HindIII
2 pUC119 control
3 1-1A control
4 pUC119 + EcoR I
5 pUC119 + Xba I
6 [1-1A]] + Spe I
7 pUC119 + Pst I
8 Empty

Restriction Enzyme (EcoR I, Pst I) Digestion of the vector

Reagent Amount
pSB1C3 4 uL
DW 10 uL
10x M Buffer 2 uL
BSA 2 uL
EcoR I 1 uL
Pst I 1 uL
Total 20 uL

Restriction Enzyme (Xba I, Pst I) Digestion of the Parts

RBS

Reagent Amount
1-2M 2.5 uL
DW 11.5 uL
10x M Buffer 2 uL
BSA 2 uL
Xba I 1 uL
Pst I 1 uL
Total 20 uL

Terminator

Reagent Amount
1-23L 1.5 uL
DW 12.5 uL
10x M Buffer 2 uL
BSA 2 uL
Xba I 1 uL
Pst I 1 uL
Total 20 uL

Restriction Enzyme (EcoR I, Spe I) Digestion of the Parts

Heat shock promotor

Reagent Amount
3-1E 5 uL
DW 9.7 uL
10x M Buffer 2 uL
BSA 2 uL
EcoR I 1 uL
Spe I 0.3 uL
Total 20 uL

RFP

Reagent Amount
1-18F 5 uL
DW 9.7 uL
10x M Buffer 2 uL
BSA 2 uL
EcoR I 1 uL
Spe I 0.3 uL
Total 20 uL
  • It was obvious after restriction enzyme activity check that Spe I was week, so additional 0.7 uL was added
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