Team:HokkaidoU Japan/Notebook/August27

From 2010.igem.org

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2.mixed solutions according to the table below
2.mixed solutions according to the table below
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3.incubated at 37C for 60min
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4.purified the two samples
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5.added 250 uL 100% EtOH to each samples
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6.centrifuged at 4C, 15000rpm for 10 min
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7.discarded supernatant
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8.put the samples into desiccator
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9.added 10 uL DW to pSB1C3 and 2 uL DW to 1-1A
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10.mixed 1 uL pSB1C3 and 2 uL 1-1A
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11.added 3 uL ligation solution and 0.5 uL T4 ligase
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12.transfered the sample to a tube of 500 uL
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13.incubated at 16C for 30 min
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->For not putting 3M CH3COONa into the solutions,we stopped operation up to this.
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=Preparation of 3 Piece Ligation=
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*Parts Information
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Heat Sensor
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BBa_K098995 3-1E 937bp
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RBS
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BBa_B0034 1-2M 12bp
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RFP
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BBa_E1010 1-18F 681bp
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Double Terminator
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BBa_0015 1-23L 129bp

Revision as of 12:40, 13 October 2010

Notebook

ラオリ英語添削お願い

Transfer 1-1A on pSB1A10 to pSB1C3

1.estimated concentration of pSB1C3 and 1-1A which was amplified by miniprep before

2.mixed solutions according to the table below


Reagent Amount
pSB1C3 5 uL
DW 75 uL
10x M Buffer 10 uL
1%BSA 10 uL
EcoRI 5 uL
PstI 5 uL
Total 100 uL
Reagent Amount
1-1A 8 uL
DW 6 uL
10x M Buffer 2 uL
1%BSA 2 uL
EcoRI 1 uL
PstI 1 uL
Total 20 uL













3.incubated at 37C for 60min

4.purified the two samples

5.added 250 uL 100% EtOH to each samples

6.centrifuged at 4C, 15000rpm for 10 min

7.discarded supernatant

8.put the samples into desiccator

9.added 10 uL DW to pSB1C3 and 2 uL DW to 1-1A

10.mixed 1 uL pSB1C3 and 2 uL 1-1A

11.added 3 uL ligation solution and 0.5 uL T4 ligase

12.transfered the sample to a tube of 500 uL

13.incubated at 16C for 30 min

->For not putting 3M CH3COONa into the solutions,we stopped operation up to this.


Preparation of 3 Piece Ligation

  • Parts Information

Heat Sensor

BBa_K098995 3-1E 937bp

RBS

BBa_B0034 1-2M 12bp

RFP

BBa_E1010 1-18F 681bp

Double Terminator

BBa_0015 1-23L 129bp
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