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Note Book => [ June / July / August / September - October ]

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) -> 4 samples (I, II, III, IV)

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)

I: 116.8ng/uL

II: 81.6ng/uL

III: 110.2ng/uL

IV: 139.5ng/uL

Inoculation

8-4 (7/23 transformation)

Sequencing

1-99234 I, II, III, IV (7/24 miniprep)

1-9234 I, II, III, IV (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): 82.4ng/uL

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)

I: 65.4ng/uL

II: 29.6ng/uL

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