Team:HokkaidoU Japan/Notebook/September2

From 2010.igem.org

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=RFPとpSB1C3，RFPとpUC119のDigestion/ Ligation/ Transformation=
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{|style="text-align:center; width: 500px"
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|-
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|style="border-bottom:1px solid #000;"|DNA
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|style="border-bottom:1px solid #000;"|濃度 (ng/uL)
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|style="border-bottom:1px solid #000;"|長さ(bp)
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|style="border-bottom:1px solid #000;"|量(uL)
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|-
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|RFP (1-5A)
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|80 ng/uL
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|1000 bp
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|26 uL
 +
|-
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|pSB1C3
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|20 ng/uL
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|2000 bp
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|35 uL
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|-
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|style="background-color:#eee;"|pUC119（cut済み）
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|style="background-color:#eee;"|10 ng/uL
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|style="background-color:#eee;"|3000 bp
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|style="background-color:#eee;"|28 uL
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|}
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==Digestion==
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{|style="text-align:center; margin-left: 30px; float: left;"
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|-
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|RFP (1-5A)
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|26 uL
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|-
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|DW
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|9 uL
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|-
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|0.1% BSA
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|5 uL
 +
|-
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|10x M Buffer
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|5 uL
 +
|-
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|EcoR I
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|2 uL
 +
|-
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|Pst I
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|3 uL
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|-
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|style="border-top:1px solid #000;"|'''Total'''
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|style="border-top:1px solid #000;"|'''50 uL'''
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|}
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{|style="text-align:center; margin-left: 30px; float: left;"
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|-
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|pSB1C3
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|33 uL
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|-
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|DW
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|2 uL
 +
|-
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|0.1% BSA
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|5 uL
 +
|-
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|10x M Buffer
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|5 uL
 +
|-
 +
|EcoR I
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|2 uL
 +
|-
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|Pst I
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|3 uL
 +
|-
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|style="border-top:1px solid #000;"|'''Total'''
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|style="border-top:1px solid #000;"|'''50 uL'''
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|}
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<div style="clear:both;"></div>
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→37℃, 60 min<br>
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→10 uLの6x Sample bufferを加え，次のように泳動
 +
 +
==電気泳動＆ゲル抽==
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レーン２～５は濃度測定のため各1 uLのDNAを泳動した．
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{|style="width: 300px"
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|-
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|1
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|λ/''Hin''d III, EcoR I
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|-
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|2
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|50 base ladder
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|-
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|3
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|Heat shock promotor
 +
|-
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|4
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|RBS
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|-
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|5
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|RFP protein coding
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|-
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|6
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|double Terminator
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|-
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|7
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|blank
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|-
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|8
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|style="border-left:1px solid #000;" rowspan="4"|RFP reporter (1-5A) 15 uL each
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|-
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|9
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|-
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|10
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|-
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|11
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|-
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|12
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|blank
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|-
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|13
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|rowspan="4" style="border-left:1px solid #000;"|pSB1C3 15 uL each
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|-
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|14
 +
|-
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|15
 +
|-
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|16
 +
|-
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|17
 +
|blank
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|}
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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．<br>
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この時点でRFP 104 ng/uL，pSB1C3 33 ng/uL．
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 +
==Ligation==
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{|style="text-align:center; margin-left: 30px; float: left;"
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|-
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|RFP
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|1.5 uL
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|-
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|pSB1C3
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|1.5 uL
 +
|-
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|ligation Kit I
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|3 uL
 +
|-
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|T4 ligase
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|0.5 uL
 +
|-
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|style="border-top:1px solid #000;"|'''Total'''
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|style="border-top:1px solid #000;"|'''6.5 uL'''
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|}
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{|style="text-align:center; margin-left: 30px; float: left;"
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|-
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|RFP
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|1.5 uL
 +
|-
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|pSB1C3
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|1.5 uL
 +
|-
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|Mighty Mix
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|3 uL
 +
|-
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|T4 ligase
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|0.5 uL
 +
|-
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|style="border-top:1px solid #000;"|'''Total'''
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|style="border-top:1px solid #000;"|'''6.5 uL'''
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|}
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{|style="text-align:center; margin-left: 30px; float: left;"
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|-
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|RFP
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|0.8 uL
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|-
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|pUC119
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|4 uL
 +
|-
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|ligation Kit I
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|4.8 uL
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|-
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|T4 ligase
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|0.5 uL
 +
|-
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|style="border-top:1px solid #000;"|'''Total'''
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|style="border-top:1px solid #000;"|'''10.5 uL'''
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|}
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{|style="text-align:center; margin-left: 30px; float: left;"
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|-
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|RFP
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|0.8 uL
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|-
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|pUC119
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|4 uL
 +
|-
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|Mighty Mix
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|4.8 uL
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|-
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|T4 ligase
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|0.5 uL
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|-
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|style="border-top:1px solid #000;"|'''Total'''
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|style="border-top:1px solid #000;"|'''10.5 uL'''
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|}
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<div style="clear:both;"></div>
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→16℃, 30 min<br>
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→Transformation
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 +
 +
=HSP, RBS, RFP, dTのゲル抽=

Revision as of 12:44, 18 September 2010

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．

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=
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September2"