Team:HokkaidoU Japan/Notebook/August16
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Latest revision as of 07:31, 27 October 2010
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
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