Team:HokkaidoU Japan/Notebook/September2
From 2010.igem.org
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Revision as of 07:07, 20 September 2010
RFPとpSB1C3,RFPとpUC119のDigestion/ Ligation/ Transformation
DNA | Concentration | Length | Amount |
---|---|---|---|
RFP (1-5A) | 80 ng/uL | 1000 bp | 26 uL |
pSB1C3 | 20 ng/uL | 2000 bp | 35 uL |
pUC119(cut済み) | 10 ng/uL | 3000 bp | 28 uL |
Digestion
Reagent | Amount |
---|---|
RFP (1-5A) | 26 uL |
DW | 9 uL |
0.1% BSA | 5 uL |
10x M Buffer | 5 uL |
EcoR I | 2 uL |
Pst I | 3 uL |
Total | 50 uL |
Reagent | Amount |
---|---|
pSB1C3 | 33 uL |
DW | 2 uL |
0.1% BSA | 5 uL |
10x M Buffer | 5 uL |
EcoR I | 2 uL |
Pst I | 3 uL |
Total | 50 uL |
→37℃, 60 min
→10 uLの6x Sample bufferを加え,次のように泳動
電気泳動&ゲル抽
レーン2~5は濃度測定のため各1 uLのDNAを泳動した.
Lane | DNA |
1 | λ/Hind III, EcoR I |
2 | 50 base ladder |
3 | Heat shock promotor |
4 | RBS |
5 | RFP protein coding |
6 | double Terminator |
7 | blank |
8 | RFP reporter (1-5A) 15 uL each |
9 | |
10 | |
11 | |
12 | blank |
13 | pSB1C3 15 uL each |
14 | |
15 | |
16 | |
17 | blank |
それぞれゲルから切り出し(210 mg,220 mg),抽出キットで20 uLにした.
この時点でRFP 104 ng/uL,pSB1C3 33 ng/uL.
Ligation
Reagent | Amount |
---|---|
RFP | 1.5 uL |
pSB1C3 | 1.5 uL |
ligation Kit I | 3 uL |
T4 ligase | 0.5 uL |
Total | 6.5 uL |
Reagent | Amount |
---|---|
RFP | 1.5 uL |
pSB1C3 | 1.5 uL |
Mighty Mix | 3 uL |
T4 ligase | 0.5 uL |
Total | 6.5 uL |
Reagent | Amount |
---|---|
RFP | 0.8 uL |
pUC119 | 4 uL |
ligation Kit I | 4.8 uL |
T4 ligase | 0.5 uL |
Total | 10.5 uL |
Reagent | Amount |
---|---|
RFP | 0.8 uL |
pUC119 | 4 uL |
Mighty Mix | 4.8 uL |
T4 ligase | 0.5 uL |
Total | 10.5 uL |
→16℃, 30 min
→Transformation