Team:HokkaidoU Japan/Notebook/August16

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Revision as of 08:30, 20 September 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

電気泳動

HokkaidoU Japan 20100816a.jpg

制限酵素処理した溶液に2 uLの6x Sample Bufferを加え，電気泳動した

Lane	DNA
１	λ/HindIII
２	pUC119 コントロール
３	1-1A コントロール
４	pUC119 + EcoR I
５	pUC119 + Xba I
６	1-1A + Spe I
７	pUC119 + Pst I
８	空き

ベクターの制限酵素処理 (EcoR I, Pst I)

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

パーツの制限酵素処理 (Xba I, Pst I)

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

パーツの制限酵素処理 (EcoR I, Spe I)

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

制限酵素の活性チェックでSpe Iの活性が低かったので，あとから0.7 uL加えた

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