Team:UT-Tokyo/Sudoku notebook
From 2010.igem.org
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Revision as of 08:37, 19 September 2010
Sudoku
Abstract Construct Experiment Notebook Result
Notebook
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, 9 plasmid (6/25 miniprep)
2 : 59.3 ng/uL SP
3 : 124.2 ng/uL XP
5 : 81.3 ng/uL SP
9 : 85.4 ng/uL SP
Gel extract
2, 3, 5, 9 plasmid (7/1 enzyme digestion)
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 : ES
11 (1st) : ES -> Judged failure by cut check → miniprep again
11 (2nd) : ES
7/3
Gel extract
2-3, 11 plasmid (7/2 enzyme digestion)
Label : 100703 X ゲル抽出
7/5
Ligation
2-3 (insert, 7/3 ES enzyme digestion) & 4 (vector, probably 6/28 gel extract, EX enzyme digestion) / 11 (insert, 7/3 ES enzyme digestion) & 7 (vector, probably 6/28 gel extract, EX enzyme digestion)
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
234 plasmid (7/5 plate)
11-7 plasmid (7/5 plate) -> failure
Miniprep
234 plasmid (7/6 plate)
234 I -> 84.9 ng/uL
234 II -> 98.9 ng/uL
234 III -> 98.1 ng/uL
234 IV -> 99.4 ng/uL
Enzyme digestion
11 (7/1 miniprep)
11 : ES
ethanol precipitation
failure
Inoculation
7 plasmid (6/23 plate)
Enzyme digestion
1, 4 (6/28 enzyme digestion -> short), 11 plasmid (7/6 miniprep)
1 : SP
4 : EX
234 : XP
Inoculation
5 plasmid (6/23 plate)
7/7
Gel extract
1, 4, 234 ,11 plasmid (7/6 enzyme digestion)
Miniprep
5, 7 plasmid (7/6 plate)
5 -> 25.5 ng/uL
7 -> 22.3 ng/uL
Enzyme digestion
5,7 (7/7 miniprep)
5 : SP
7 : EX
Gel extract
5, 7 plasmid (7/7 enzyme digestion)
Ligation
1 (7/7 gel extract) & 234 (7/7 gel extract) / 9 (7/1 gel extract) & 234 (7/7 gel extract) / 7 (7/7 gel extract) & 11 (7/7 gel extract)
7/8
Enzyme digestion
11 (7/7 gel extract ,4.1 ng/uL), 7
11 : ES
7 : EX
Gel extract
7 (7/6 enzyme digestion), 234 (7/7 enzyme digestion) plasmid
Ligation
234(7/8 gel extract) & 9(7/1 gel extract)
234 : 10.2 ng/uL -> 4.1 uL & 9 : 10.6 ng/uL -> 5.9 uL
7/9
Transformation
9-234 plasmid (7/8 ligation)
Gel extract
11 plasmid (7/8 enzyme digestion)
Cut check
7 (7/8 enzyme digestion)
Inoculation
1-234 plasmid (7/7 transformation), 9 plasmid (6/23)
Ligation
11 (7/1 ligation) & 7 (7/8 ligation)
11 : 6.5 ng/uL
7/10
Miniprep
1-234 (7/9 inoculation)
1-234 : 107.2 ng/uL
Transformation
9-234
Inoculation
9 (6/23 transformation)
7/11
enzyme digestion
7 (7/7 miniprep), 234 (7/6 miniprep)
7 : EX
234 : XP
Inoculation
9-234 (7/9 transformation -> failure), 11-7 (7/10 transformation), 9 (6/23 transformation), 11 (6/30 transformation)
7/12
Miniprep
11-7, 9, 11 (7/11 inoculation)
11-7 : 81.2 ng/uL
9 : 84.4 ng/uL
11 : 81.2 ng/uL
Enzyme digestion
11-7, 9 (7/12 miniprep)
11-7 : XP
9 : SP
Cut check
1-234 (7/10 107.2 ng/uL), 234 (7/6 98.1 ng/uL), 2-3 (7/2 105.0 ng/uL), 3 (6/25 124.2 ng/uL) plasmid
7/13
Gel extraction
11-7, 9 (7/12 enzyme digestion), 2-3-4 (7/11 enzyme digestion)
Ligation
9 & 2-3-4 (7/13 gel extract)/ 5 (7/7 gel extract) & 11-7 (7/13 gel extract)
7/14
Transformation
5-11-7, 9-234 (7/13 gel extract)
Inoculation
5-11-7, 9-234 (7/14 transformation)
7/15
Miniprep
5-11-7, 9-234 (7/14 inoculation)
5-11-7 I 62.6 ng/uL
5-11-7 II 82.6 ng/uL
5-11-7 III 59.5 ng/uL
9-234 I 46.4 ng/uL
9-234 II 50.6 ng/uL
9-234 III 41.8 ng/uL
Enzyme digestion
5-11-7, 9-234 (7/15 miniprep)
7/16
Inoculation
9-234 (7/14 plate)
Ligation
9 & 234 (7/13 gel extract) / 1 (7/7 gel extract) & 5-11-7 (7/16 gel extract)
Enzyme digestion
9 (7/12 miniprep)
9 : SP
Gel extraction
9-234, 5-11-7 (7/15 enzyme digestion)
7/17
Miniprep
9-234 (7/16 inoculation)
9-234 I 35.0 ng/uL
9-234 II 75.4 ng/uL
Enzyme digestion
9-234 (7/17 miniprep)
Gel extraction
9 (7/16 enzyme digestion)
Transformation
9-234, 1-5-11-7, 9 (7/16 ligation)
7/18
Gel extract
9-234 (7/17 enzyme digestion)
Ligation
9 (vector, 7/17 SP gel extract) & 234 (insert, 7/13 XP gel extract)
9 : 10.8 ng/uL -> 5 uL & 234 : 7.1 ng/uL -> 5 uL
Ligation
9 (vector, 7/17 SP gel extract) & 9234 (insert, 7/18 XP gel extract) / 1 (vector, 7/7 SP gel extract) & 9234 (insert, 7/18 XP gel extract)
9 : 10.8 ng/uL -> 5 uL & 9234 : 8.6 ng/uL -> 5 uL
1 : 28.4 ng/uL -> 3 uL & 9234 : 8.6 ng/uL -> 7 uL
Gel extract
9-234 (7/17 XP enzyme digestion)
7/19
Transformation
9-9234, 1-9234 (7/18 ligation)
Miniprep
1-5-11-7 (7/17 inoculation)
1-5-11-7 I : 142.1 ng/uL
1-5-11-7 II : 143.9 ng/uL
1-5-11-7 III : 131.5 ng/uL
7/20
Sequencing
9-234 II(7/17 miniprep)
1-234 (7/10 miniprep)
234 (7/6 miniprep)
1-5-11-7 (7/19 miniprep)
Inoculation
9-9234, 1-9234 (7/19 plate)
7/21
Miniprep
1-9234, 9-9234 (7/20 inoculation)
1-9234 I 69.4 ng/uL
1-9234 II 58.7 ng/uL
1-9234 III 50.5 ng/uL
1-9234 IV 68.8 ng/uL
9-9234 I 100.3 ng/uL
9-9234 II 29.4 ng/uL
9-9234 III 76.8 ng/uL
9-9234 IV 70.3 ng/uL
Enzyme digestion
9-9234 I,III (7/21 miniprep) : XPcut
7/22
Gel extract
99234 plasmid (7/21 enzyme digestion)
Ligation
1 (7/7 gel extract) & 99234 (7/22 gel extract)
1: 28.4ng/uL -> 3uL, 99234: 13.2ng/uL -> 7uL
Transformation
1-99234 (7/22 ligation)
7/23
Inoculation
1-99234 (7/22 transformation)
Cut check
XScut
234 (7/6 miniprep)
9234 (7/17 miniprep)
19234 (7/21 miniprep)
99234 (7/21 miniprep)
Ligation
8 (lox 71) & 4 (7/7 gel extract)
8: 1105ng/uL -> 0.5uL, 4: 11.9ng/uL -> 4.5uL
Transformation
8-4 (7/23 ligation)
7/24
Miniprep
1-99234 (7/23 Inoculation)
Inoculation
8-4 (7/23 transformation)
Sequencing
1-99234 (7/24 miniprep)
1-9234 (7/21 miniprep)
PCR program
1. 96℃, 1 minute
2. 98℃, 10 seconds
3. 50℃, 5 seconds
4. 60℃, 4minutes
5. 2,3,4 x 25
6. 4℃, forever
7/25
Enzyme digestion
8-4 (7/25 miniprep): XScut, EXcut
4 (control): XScut
1-5-11-7 (7/14 miniprep): EScut
Miniprep
8-4 (7/24 inoculation)
7/26
Gel extract
8-4 EXcut (7/25 enzyme digestion)
1-5-11-7 EScut (7/25 enzyme digestion)
Cut check
8-4 XScut (7/25 enzyme digestion) -> failed
4 XScut (7/25 enzyme digestion)
Ligation
8 (7/23 annealing) & 4 (7/7 gel extraction)
8: 200ng/uL -> 0.5uL, 4: 11.9ng/uL -> 4.5uL
Transformation
8-4 (7/26 ligation)
7/27
Ligation
1-5-11-7 (7/26 gel extraction) & 8-4 (7/26 gel extraction)
1-5-11-7 (EScut): 13.1ng/uL -> 4.4uL, 8-4 (EXcut): 12.5ng/uL -> 5.6uL
Transformation
1-5-11-7-8-4 (7/27 ligation)
Inoculation
8-4 (7/26 transformation)
Miniprep
8-4 (7/27 inoculation)
Enzyme digestion
8-4 (7/27 miniprep): EScut
4 (6/25 miniprep): EScut
7/28
Gel extract
8-4 (7/27 enzyme digestion)
Ligation
1-5-11-7 (7/26 gel extraction) & 8-4 (7/28 gel extraction)
1-5-11-7 (EScut): 13.1ng/uL -> 5uL, 8-4 (EXcut): 12.5ng/uL -> 5uL
Inoculation
1-5-11-7-8-4 (7/27 transformation) -> 4 samples (I, II, III, IV)
7/29
Miniprep
1-5-11-7-8-4 I, II, III, IV (7/28 inoculation)
I 109.8ng/uL
II 100.7ng/uL
III 118.9ng/uL
IV 118.0ng/uL
Transformation
1-5-11-7-8-4 I, II, III, IV (7/28 miniprep)
Cut check
1-5-11-7 (control)
1-5-11-7-8-4 I
1-5-11-7-8-4 II
1-5-11-7-8-4 III
1-5-11-7-8-4 IV -> failed
Sequencing
1-5-11-7-8-4 I, II, III
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)