Team:UT-Tokyo/Sudoku notebook

From 2010.igem.org

(Difference between revisions)
(Pre-experiment)
 
(30 intermediate revisions not shown)
Line 1: Line 1:
-
{{UT-Tokyo_HEADER}}
+
__NOTOC__{{UT-Tokyo_CSS3}}{{UT-Tokyo_CSS_p}}{{UT-Tokyo_Head}}
-
<div id="main_all">
+
= '''Sudoku''' =
= '''Sudoku''' =
-
 
+
<html>
-
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_abstract  Abstract]   
+
<div>
-
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_construct Construct]     
+
        <ul id="inpagemenu">
-
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_experiment Experiment]     
+
                <li><a href="/Team:UT-Tokyo/Sudoku_abstract" id="abstract">Introduction</a></li>
-
Notebook   
+
                <li><a href="/Team:UT-Tokyo/Sudoku_construct" id="construct">System</a></li>
-
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_result Result]
+
                <li><a href="/Team:UT-Tokyo/Sudoku_modeling" id="modeling">Modeling</a></li>
 +
                <li><span>Experiments</span></li>
 +
                <li><a href="/Team:UT-Tokyo/Sudoku_perspective" id="perspective">Perspective</a></li>
 +
            <li><a href="/Team:UT-Tokyo/Sudoku_reference" id="reference">Reference</a></li>
 +
            </ul>
 +
</div>
 +
<div id="clear"></div>
 +
</html><br />
 +
Note Book =>
 +
[
 +
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook_6 June]    /
 +
July    /
 +
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook_2 August]    /
 +
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook_8 September - October]   
 +
]
== '''Notebook''' ==
== '''Notebook''' ==
Line 22: Line 35:
2-3 plasmid (6/30 ligation)
2-3 plasmid (6/30 ligation)
-
< 6/30 transformation fail >
+
< 6/30 transformation -> fail >
Line 32: Line 45:
'''Enzyme digestion'''
'''Enzyme digestion'''
-
2, 3, 5, 7 plasmid (6/25 miniprep)
+
2, 3, 5, 9 plasmid (6/25 miniprep)
-
2 : 53.9 ng/uL → about 15 uL (shortage) / H / SP (2 uL)
+
2 : 59.3 ng/uL SP
-
3 : 124.2 ng/uL → 8 uL / M / XP (2 uL)
+
3 : 124.2 ng/uL XP
-
5 : 81.3 ng/uL → 12.3 uL / H / SP (2 uL)
+
5 : 81.3 ng/uL SP
-
7 : 85.4 ng/uL → 11.7 uL / H / SP (2 uL)
+
9 : 85.4 ng/uL SP
'''Gel extract'''
'''Gel extract'''
-
2, 3, 5, 7 plasmid (7/1 enzyme cut)
+
2, 3, 5, 9 plasmid (7/1 enzyme digestion)
Label : 100701 数X (2,3,5,9) ゲル抽出
Label : 100701 数X (2,3,5,9) ゲル抽出
Line 57: Line 70:
2-3, 11 plasmid (7/1 inoculation)
2-3, 11 plasmid (7/1 inoculation)
-
2-3 105 ng/uL
+
2-3 -> 105 ng/uL
-
11 (1st) 191.3 ng/uL
+
11 (1st) -> 191.3 ng/uL
-
11 (2nd) 49.5 ng/uL
+
11 (2nd) -> 49.5 ng/uL
Line 68: Line 81:
2-3, 11 plasmid (7/2 miniprep)
2-3, 11 plasmid (7/2 miniprep)
-
2-3 : 9.5 uL / H / ES (1.5 uL)
+
2-3 : ES
-
11 (1st) : 5.2 uL / H /  ES (1.5 uL) → Judged failure by cut check → miniprep again
+
11 (1st) : ES -> Judged failure by cut check → miniprep again
-
11 (2nd) : 20.2 uL / H /  ES (1.5 uL)
+
11 (2nd) : ES
Line 80: Line 93:
'''Gel extract'''
'''Gel extract'''
-
2-3, 11 plasmid (7/2 enzyme cut)
+
2-3, 11 plasmid (7/2 enzyme digestion)
Label : 100703 X ゲル抽出
Label : 100703 X ゲル抽出
Line 90: Line 103:
'''Ligation'''
'''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 (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
+
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
+
11 : 4.2 ng/uL -> 7.5 uL & 7 : Unknown -> 2.5 uL
Line 100: Line 113:
2-3-4 plasmid (7/5 ligation) / 11-7 plasmid (7/5 ligation)
2-3-4 plasmid (7/5 ligation) / 11-7 plasmid (7/5 ligation)
-
 
Line 107: Line 119:
'''Inoculation'''
'''Inoculation'''
-
5 plasmid (6/23 plate)
+
234 plasmid (7/5 plate)
 +
11-7 plasmid (7/5 plate) -> failure
-
'''Enzyme digestion'''
 
-
1, 4 (6/28 enzyme digest → short), 11 plasmid (7/6 miniprep)
+
'''Miniprep'''
 +
 
 +
234 plasmid (7/6 plate)
 +
 
 +
234 I -> 84.9 ng/uL
-
1 : 8.5 uL / H / SP (1.5 uL)
+
234 II -> 98.9 ng/uL
-
4 : 8.7 uL / M /  EX (1.5 uL)
+
234 III -> 98.1 ng/uL
-
234 : 10 uL / M / XP (1.5 uL)
+
234 IV -> 99.4 ng/uL
Line 125: Line 141:
11 (7/1 miniprep)
11 (7/1 miniprep)
-
11 : 5.2 uL / H / ES (EcoRI: 1.5 uL, SpeI: 1 uL)
+
11 : ES
Line 136: Line 152:
7 plasmid (6/23 plate)
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'''
'''Inoculation'''
-
234 plasmid (7/5 plate)
+
5 plasmid (6/23 plate)
-
11-7 plasmid ()
 
-
=== Check test ===
 
-
== Main construct ==
 
-
=== Research of SP6 RNApol cDNA and F plasmid ===
+
==== 7/7 ====
 +
'''Gel extract'''
 +
1, 4, 234 ,11 plasmid (7/6 enzyme digestion)
-
=== Convert lox71(rev) to lox66(rev) ===
 
 +
'''Miniprep'''
 +
5, 7 plasmid (7/6 plate)
-
=== Make Hin(rev) ===
+
5 -> 25.5 ng/uL
 +
7 -> 22.3 ng/uL
-
=== Revise MS2 gene ===
+
'''Enzyme digestion'''
 +
5,7 (7/7 miniprep)
 +
5 : SP
-
=== Make location(rev)-rbs(rev)-location(rev) ===
+
7 : EX
 +
'''Gel extract'''
 +
5, 7 plasmid (7/7 enzyme digestion)
-
</div>
+
 
-
{{UT-Tokyo_FOOTER}}
+
'''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
 +
 
 +
{{UT-Tokyo_Foot}}

Latest revision as of 02:59, 28 October 2010

UT-Tokyo

Sudoku


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