Team:HokkaidoU Japan/Notebook/September2
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+ | =RFPとpSB1C3,RFPとpUC119のDigestion/ Ligation/ Transformation= | ||
+ | {|style="text-align:center; width: 500px" | ||
+ | |- | ||
+ | |style="border-bottom:1px solid #000;"|DNA | ||
+ | |style="border-bottom:1px solid #000;"|濃度 (ng/uL) | ||
+ | |style="border-bottom:1px solid #000;"|長さ(bp) | ||
+ | |style="border-bottom:1px solid #000;"|量(uL) | ||
+ | |- | ||
+ | |RFP (1-5A) | ||
+ | |80 ng/uL | ||
+ | |1000 bp | ||
+ | |26 uL | ||
+ | |- | ||
+ | |pSB1C3 | ||
+ | |20 ng/uL | ||
+ | |2000 bp | ||
+ | |35 uL | ||
+ | |- | ||
+ | |style="background-color:#eee;"|pUC119(cut済み) | ||
+ | |style="background-color:#eee;"|10 ng/uL | ||
+ | |style="background-color:#eee;"|3000 bp | ||
+ | |style="background-color:#eee;"|28 uL | ||
+ | |} | ||
+ | |||
+ | ==Digestion== | ||
+ | {|style="text-align:center; margin-left: 30px; float: left;" | ||
+ | |- | ||
+ | |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 | ||
+ | |- | ||
+ | |style="border-top:1px solid #000;"|'''Total''' | ||
+ | |style="border-top:1px solid #000;"|'''50 uL''' | ||
+ | |} | ||
+ | |||
+ | {|style="text-align:center; margin-left: 30px; float: left;" | ||
+ | |- | ||
+ | |pSB1C3 | ||
+ | |33 uL | ||
+ | |- | ||
+ | |DW | ||
+ | |2 uL | ||
+ | |- | ||
+ | |0.1% BSA | ||
+ | |5 uL | ||
+ | |- | ||
+ | |10x M Buffer | ||
+ | |5 uL | ||
+ | |- | ||
+ | |EcoR I | ||
+ | |2 uL | ||
+ | |- | ||
+ | |Pst I | ||
+ | |3 uL | ||
+ | |- | ||
+ | |style="border-top:1px solid #000;"|'''Total''' | ||
+ | |style="border-top:1px solid #000;"|'''50 uL''' | ||
+ | |} | ||
+ | <div style="clear:both;"></div> | ||
+ | →37℃, 60 min<br> | ||
+ | →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. | ||
+ | {|style="width: 300px" | ||
+ | |- | ||
+ | |1 | ||
+ | |λ/''Hin''d III, EcoR I | ||
+ | |- | ||
+ | |2 | ||
+ | |50 base ladder | ||
+ | |- | ||
+ | |3 | ||
+ | |Heat shock promotor | ||
+ | |- | ||
+ | |4 | ||
+ | |RBS | ||
+ | |- | ||
+ | |5 | ||
+ | |RFP protein coding | ||
+ | |- | ||
+ | |6 | ||
+ | |double Terminator | ||
+ | |- | ||
+ | |7 | ||
+ | |blank | ||
+ | |- | ||
+ | |8 | ||
+ | |style="border-left:1px solid #000;" rowspan="4"|RFP reporter (1-5A) 15 uL each | ||
+ | |- | ||
+ | |9 | ||
+ | |- | ||
+ | |10 | ||
+ | |- | ||
+ | |11 | ||
+ | |- | ||
+ | |12 | ||
+ | |blank | ||
+ | |- | ||
+ | |13 | ||
+ | |rowspan="4" style="border-left:1px solid #000;"|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.<br> | ||
+ | この時点でRFP 104 ng/uL,pSB1C3 33 ng/uL. | ||
+ | |||
+ | ==Ligation== | ||
+ | {|style="text-align:center; margin-left: 30px; float: left;" | ||
+ | |- | ||
+ | |RFP | ||
+ | |1.5 uL | ||
+ | |- | ||
+ | |pSB1C3 | ||
+ | |1.5 uL | ||
+ | |- | ||
+ | |ligation Kit I | ||
+ | |3 uL | ||
+ | |- | ||
+ | |T4 ligase | ||
+ | |0.5 uL | ||
+ | |- | ||
+ | |style="border-top:1px solid #000;"|'''Total''' | ||
+ | |style="border-top:1px solid #000;"|'''6.5 uL''' | ||
+ | |} | ||
+ | |||
+ | {|style="text-align:center; margin-left: 30px; float: left;" | ||
+ | |- | ||
+ | |RFP | ||
+ | |1.5 uL | ||
+ | |- | ||
+ | |pSB1C3 | ||
+ | |1.5 uL | ||
+ | |- | ||
+ | |Mighty Mix | ||
+ | |3 uL | ||
+ | |- | ||
+ | |T4 ligase | ||
+ | |0.5 uL | ||
+ | |- | ||
+ | |style="border-top:1px solid #000;"|'''Total''' | ||
+ | |style="border-top:1px solid #000;"|'''6.5 uL''' | ||
+ | |} | ||
+ | |||
+ | {|style="text-align:center; margin-left: 30px; float: left;" | ||
+ | |- | ||
+ | |RFP | ||
+ | |0.8 uL | ||
+ | |- | ||
+ | |pUC119 | ||
+ | |4 uL | ||
+ | |- | ||
+ | |ligation Kit I | ||
+ | |4.8 uL | ||
+ | |- | ||
+ | |T4 ligase | ||
+ | |0.5 uL | ||
+ | |- | ||
+ | |style="border-top:1px solid #000;"|'''Total''' | ||
+ | |style="border-top:1px solid #000;"|'''10.5 uL''' | ||
+ | |} | ||
+ | |||
+ | {|style="text-align:center; margin-left: 30px; float: left;" | ||
+ | |- | ||
+ | |RFP | ||
+ | |0.8 uL | ||
+ | |- | ||
+ | |pUC119 | ||
+ | |4 uL | ||
+ | |- | ||
+ | |Mighty Mix | ||
+ | |4.8 uL | ||
+ | |- | ||
+ | |T4 ligase | ||
+ | |0.5 uL | ||
+ | |- | ||
+ | |style="border-top:1px solid #000;"|'''Total''' | ||
+ | |style="border-top:1px solid #000;"|'''10.5 uL''' | ||
+ | |} | ||
+ | <div style="clear:both;"></div> | ||
+ | →16℃, 30 min<br> | ||
+ | →Transformation | ||
+ | |||
+ | |||
+ | =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