Team:UT-Tokyo/Sudoku notebook 6

From 2010.igem.org

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(Notebook)
(Notebook)
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'''Transformation'''
'''Transformation'''
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2010 Kit plate1
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2010 Kit plate1: 6N,1H,5D,17J,4H,18A
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6N,1H,5D,17J,4H,18A
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2010 Kit plate2: 24C,2K
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2010 Kit plate2
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24C,2K
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'''Inoculation'''
'''Inoculation'''
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2010 Kit plate1
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2010 Kit plate1: 6N,1H,5D,17J,4H,18A
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6N,1H,5D,17J,4H,18A
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2010 Kit plate2
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24C,2K
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2010 Kit plate2: 24C,2K

Revision as of 05:13, 19 September 2010

Sudoku

Abstract Construct Experiment Notebook Result

Notebook

6/23

Transformation

2010 Kit plate1: 6N,1H,5D,17J,4H,18A

2010 Kit plate2: 24C,2K


6/24

Inoculation

2010 Kit plate1: 6N,1H,5D,17J,4H,18A

2010 Kit plate2: 24C,2K


6/25

Enxyme digestion

1~7, 9 & 10 plasmid

1: 117.2 ng/uL -> 2.56 uL / SP

2: 59.3 ng/uL -> 5.06 uL / SP

3: 124.2 ng/uL -> 2.42 uL / XP

4: 114.5 ng/uL -> 2.62 uL / EX

5: 81.3 ng/uL -> 3.69 uL / SP

6: 101.7 ng/uL -> 2.95 uL / ES

7: 81.5 ng/uL -> 3.68 uL / EX

9: 85.4 ng/uL -> 3.51 uL / SP

10: 73.5 ng/uL -> 4.08 uL / SP


6/28

Gel extract

1~7, 9 & 10 plasmid (6/25 enzyme digestion)

6 -> failed, others -> succeeded


6/29

Cutcheck

EScut: 1~5, 7, 9 & 10 (6/25 enzyme digestion)

HindIII cut: 6


6/30

Ligation

Transformation

Pre-experiment

Make construct

7/1

Transformation

2-3 plasmid (6/30 ligation)

< 6/30 transformation → fail >


Inoculation

11 plasmid (6/30 thaw, transformation)


Enzyme digestion

2, 3, 5, 7 plasmid (6/25 miniprep)

2 : 53.9 ng/uL → about 15 uL (shortage) / H / SP (2 uL)

3 : 124.2 ng/uL → 8 uL / M / XP (2 uL)

5 : 81.3 ng/uL → 12.3 uL / H / SP (2 uL)

7 : 85.4 ng/uL → 11.7 uL / H / SP (2 uL)


Gel extract

2, 3, 5, 7 plasmid (7/1 enzyme cut)

Label : 100701 数X (2,3,5,9) ゲル抽出


7/2

Miniprep

2-3, 11 plasmid (7/1 inoculation)

2-3 → 105 ng/uL

11 (1st) → 191.3 ng/uL

11 (2nd) → 49.5 ng/uL


Enzyme digestion

2-3, 11 plasmid (7/2 miniprep)

2-3 : 9.5 uL / H / ES (1.5 uL)

11 (1st) : 5.2 uL / H / ES (1.5 uL) → Judged failure by cut check → miniprep again

11 (2nd) : 20.2 uL / H / ES (1.5 uL)


7/3

Gel extract

2-3, 11 plasmid (7/2 enzyme cut)

Label : 100703 X ゲル抽出


7/5

Ligation

2-3 (insert, 7/3 ES enzyme cut) & 4 (vector, probably 6/28 gel extract, EX enzyme cut) / 11 (insert; 7/3 ES enzyme cut) & 7 (vector, probably 6/28 gel extract, EX enzyme cut)

2-3 : 37.5 ng/uL → 5 uL & 4 : 5.2 ng/uL → 5 uL

11 : 4.2 ng/uL → 7.5 uL & 7 : Unknown → 2.5 uL


Transformation

2-3-4 plasmid (7/5 ligation) / 11-7 plasmid (7/5 ligation)


7/6

Inoculation

5 plasmid (6/23 plate)


Enzyme digestion

1, 4 (), 11 plasmid (7/6 miniprep)

2-3 : 9.5 uL / H / ES (1.5 uL)

11 (1st) : 5.2 uL / H / ES (1.5 uL) → Judged failure by cut check → miniprep again

11 (2nd) : 20.2 uL / H / ES (1.5 uL)

Check test

Main construct

Research of SP6 RNApol cDNA and F plasmid

Convert lox71(rev) to lox66(rev)

Make Hin(rev)

Revise MS2 gene

Make location(rev)-rbs(rev)-location(rev)

Copyright © 2010 iGEM UT-Tokyo. All rights reserved.