Team:HokkaidoU Japan/Notebook/September2

From 2010.igem.org

(Difference between revisions)
(→Ligation)
(→RFPとpSB1C3，RFPとpUC119のDigestion/ Ligation/ Transformation)
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|35 uL
|35 uL
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|-
-
|style="background-color:#eee;"|pUC119（cut済み）
+
|pUC119（cut済み）
-
|style="background-color:#eee;"|10 ng/uL
+
|10 ng/uL
-
|style="background-color:#eee;"|3000 bp
+
|3000 bp
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|style="background-color:#eee;"|28 uL
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|28 uL
|}
|}

Revision as of 06:30, 19 September 2010

Notebook

RFPとpSB1C3，RFPとpUC119のDigestion/ Ligation/ Transformation

DNA 濃度 (ng/uL) 長さ(bp) 量(uL)
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

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
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．

HokkaidoU Japan 20100902a.jpg
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

RFP 1.5 uL
pSB1C3 1.5 uL
ligation Kit I 3 uL
T4 ligase 0.5 uL
Total 6.5 uL
RFP 1.5 uL
pSB1C3 1.5 uL
Mighty Mix 3 uL
T4 ligase 0.5 uL
Total 6.5 uL
RFP 0.8 uL
pUC119 4 uL
ligation Kit I 4.8 uL
T4 ligase 0.5 uL
Total 10.5 uL
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

=HSP, RBS, RFP, dTのゲル抽=
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