Team:UT-Tokyo/Sudoku notebook 2

From 2010.igem.org

(Difference between revisions)
(→Make construct)
 
(41 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>
-
== '''Notebook''' ==
+
                <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>
-
== Pre-experiment ==
+
            </ul>
-
 
+
</div>
-
AUGUST!!!!!
+
<div id="clear"></div>
-
 
+
</html><br />
-
 
+
Note Book =>
-
 
+
[
-
AUGUST!!!!!
+
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook_6 June]    /
-
 
+
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook July]    /
-
 
+
August     /
-
 
+
[https://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook_8 September - October]  
-
 
+
]
-
AUGUST!!!!!
+
-
AUGUST!!!!!
+
-
 
+
-
 
+
-
AUGUST!!!!!
 
-
=== Make construct ===
+
== Note Book ==
Line 47: Line 41:
'''Enzyme digestion'''
'''Enzyme digestion'''
-
i  1-99234Ⅰ EScut (7/24 Miniprep & Sequencing)
+
EScut  i  1-99234I  (7/24 Miniprep & Sequencing)
-
ii  1-9234Ⅰ EScut (7/21 Miniprep / 7/24 Sequencing)
+
EScut  ii  1-9234I  (7/21 Miniprep / 7/24 Sequencing)
-
iii 1,5,11,7,8,4Ⅱ EXcut (7/29 Miniprep)　
+
EXcut  iii 1,5,11,7,8,4II  (7/29 Miniprep)　
Line 65: Line 59:
'''Ligation'''
'''Ligation'''
-
i-iii i:6uL iii:6uL ligationmix 12uL
+
i-iii, ii-iii
-
 
+
-
ii-iii ii:6uL iii:4uL ligationmix 10uL
+
(8/4 Miniprep)
(8/4 Miniprep)
Line 89: Line 81:
'''Enzyme digestion'''
'''Enzyme digestion'''
-
1234（107.2ng/uL）(8/4 gel extract)
+
1234  107.2ng/uL (8/4 gel extract)
Line 115: Line 107:
'''Inoculation'''
'''Inoculation'''
-
0-iii,(i-iii),ii-iii (8/5 transformation)
+
0-iii, (i-iii), ii-iii (8/5 transformation)
Line 192: Line 184:
sudoku iii-II(7/29)
sudoku iii-II(7/29)
 +
 +
 +
==== 8/13 ====
 +
 +
'''colonyPCR'''
 +
 +
sudoku 0-iii (x16)
 +
 +
program PCR
 +
 +
94℃ 10min
 +
 +
{94℃ 20sec 53℃ 20sec 72℃ 3min}x35
 +
 +
4℃ forever
 +
 +
 +
'''Sequencing(ethanol precipitation)'''
 +
 +
master mix(8/13)
 +
 +
 +
==== 8/14 ====
 +
 +
'''Miniprep'''
 +
 +
iii(II)-Ⅰ  113.4 ng/uL
 +
 +
iii(II)-II  98.9 ng/uL
 +
 +
iii(II)-III 153.2 ng/uL
 +
 +
(8/13 Inoculation)
 +
 +
 +
==== 8/16 ====
 +
 +
'''Enzyme digestion'''
 +
 +
EXcut 1,12
 +
 +
EScut 4
 +
 +
SPcut 12
 +
 +
XPcut 2,3,4
'''Gel extract'''
'''Gel extract'''
-
0-iii I 20uL
+
1,2,3,4,12
-
0-iii II 20uL
 
-
0-iii III 20uL
+
'''ligation'''
 +
 
 +
12-2,3,4
 +
 
 +
 
 +
'''Inoculation'''
 +
 
 +
4,14
 +
 
 +
 
 +
'''transformation'''
 +
 
 +
12-2,3,4
 +
 
 +
 
 +
==== 8/18 ====
 +
 
 +
'''Gel extract'''
 +
 
 +
4-1 EXcut  20.6ng/uL
 +
 
 +
4-2 EScut  4.3ng/uL
 +
 
 +
(8/17 enzyme digestion)
 +
 
 +
 
 +
'''Inoculation'''
 +
 
 +
15 (8/17 transformation)
 +
 
 +
 
 +
==== 8/19 ====
 +
 
 +
'''Miniprep'''
-
0-iii IV 20uL
+
15-I, 15-II (8/18 Inoculation)
-
SEG 1 XPcut 20uL
+
15-I　75.3ng/uL
-
SEG 1 EScut 20uL
+
15-II　74.8ng/uL
'''Ligation'''
'''Ligation'''
-
linker EXS +Omp A
+
12 EXcut  21.6ng/uL (8/16 enzyme digestion & gel extract)
-
linker EXP +Cryj1
+
4-I EScut  4.3ng/uL (8/17 enzyme digestion & 8/18 gel extract)
-
linker EXP + Cryj2
+
4-I EXcut  ?ng/uL  (8/17 enzyme digestion & 8/18 gel extract)
 +
4-12
-
==== 8/13 ====
+
vector 12EX　7.7ul
-
'''colonyPCR'''
+
insert 4ES  2.3ul
-
sudoku 0-iii (x16)
+
4-4
-
program PCR
+
vector 4EX  8uL
-
94℃ 10min
+
insert 4ES  2uL
-
{94℃ 20sec 53℃ 20sec 72℃ 3min}×35
 
-
4℃ forever
+
'''Transformation'''
 +
4-1, 4-12, 4-4 (8/19 ligation)
-
'''Sequencing(ethanol precipitation)'''
+
12-234  (8/16 ligation)
-
master mix(8/13)
 
 +
'''Enzyme digestion'''
-
==== 8/14 ====
+
SPcut  12-I (8/4 Miniprep)
 +
==== 8/20 ====
 +
'''Enzyme digestion'''
-
==== 8/12 ====
+
EXcut  12-I (8/4 Miniprep)
-
==== 8/12 ====
+
-
==== 8/12 ====
+
-
==== 8/12 ====
+
 +
'''Ligation'''
 +
12-I  SPcut  10.2ng/uL, 2000bp (8/20 gel extract) &
 +
2,3,4  XPcut  10.2ng/uL, 1100bp (7/8 gel extract)
-
=== Check test ===
+
12-I  SPcut  10.2ng/uL (8/20 gel extract) &
 +
2,3,4  XPcut  9.1ng/uL (8/16 enzyme digestion & gel extract)
 +
'''Transformation'''
 +
12-2,3,4 (8/20 ligation) x2
 +
==== 8/21 ====
-
== Main construct ==
+
'''Miniprep'''
-
=== Research of SP6 RNApol cDNA and F plasmid ===
+
4-4 (I, II) (8/20 inoculation)
 +
'''Annealing'''
-
=== Convert lox71(rev) to lox66(rev) ===
+
PSP6revES（17） f & r  232.2ng/uL
 +
Lox2272XP（18） f & r  476.3ng/uL
 +
loadingrevXP（19） f & r  363.8ng/uL
-
=== Make Hin(rev) ===
 
 +
'''Inoculation'''
 +
sudoku1 & 4-4
-
=== Revise MS2 gene ===
 
 +
'''Ligation'''
 +
pSP6revES(17)  1uL  (vector)
-
=== Make location(rev)-rbs(rev)-location(rev) ===
+
lox2272XP(18) 1uL  (vector)
 +
loading revXP(19)  1uL  (vector)
 +
4-I EScut 4.3ng/uL, 95bp  (8/19 enzyme digestion)
 +
12 EXcut 21.6ng/uL, 2000bp  (8/16 enzyme digestion)
-
</div>
+
 
-
{{UT-Tokyo_FOOTER}}
+
'''Transformation'''
 +
 
 +
4-1  (8/19 ligation)
 +
 
 +
4-12  (8/21 ligation)
 +
 
 +
 
 +
'''Gel extant'''
 +
 
 +
12-I  (8/19 enzyme digestion)
 +
 
 +
 
 +
'''RT-PCR'''
 +
 
 +
cDNA of MS2
 +
 
 +
PCR program
 +
 
 +
1. 94℃, 2 minutes
 +
 
 +
2. 94℃, 15 seconds
 +
 
 +
3. 55℃, 30 seconds
 +
 
 +
4. 68℃, 3minutes 3 seconds
 +
 
 +
5. 2,3,4 x 24
 +
 
 +
6. 68℃, 4 minutes
 +
 
 +
7. 4℃, forever
 +
 
 +
 
 +
==== 8/22 ====
 +
 
 +
'''purification'''
 +
 
 +
cDNA of MS2 (8/21 PCR)
 +
 
 +
 
 +
'''Miniprep'''
 +
 
 +
1(II)
 +
 
 +
I　99.2ng/uL
 +
 
 +
II  112.8ng/uL
 +
 
 +
(8/21 Inoculation)
 +
 
 +
4-4
 +
 
 +
III　45.6ng/uL
 +
 
 +
IV　33.4ng/uL
 +
 
 +
(8/21 Inoculation)
 +
 
 +
12-2,3,4
 +
 
 +
I　17.9ng/uL
 +
 
 +
II　3.3ng/uL
 +
 
 +
(7/8 ligation & transformation / 8/21 Inoculation)
 +
 
 +
III　1.1ng/uL
 +
 
 +
IV　7.3ng/uL
 +
 
 +
(8/16 ligation & transformation / 8/21 Inoculation)
 +
 
 +
 
 +
'''Inoculation'''
 +
 
 +
4-4 & 12-234
 +
 
 +
(8/22 Miniprep)
 +
 
 +
 
 +
'''XPcut(RT-PCR)'''
 +
 
 +
XPcut cDNA of MS2 with Restriction Enzyme sites
 +
 
 +
(8/22 purification)
 +
 
 +
 
 +
'''Gel extract'''
 +
 
 +
12-I EXcut  12.3ng/uL
 +
(8/20 enzyme digestion)
 +
 
 +
 
 +
'''Transformation'''
 +
 
 +
17, 18, 19  (8/21 ligation)
 +
 
 +
 
 +
'''Enzyme digestion'''
 +
 
 +
1(II)-II EXcut  (8/22  miniprep)
 +
 
 +
 
 +
'''Ligation'''
 +
 
 +
12-I EXcut  (8/22 gel extract)
 +
 
 +
4-I EScut  (8/18 gel extract)
 +
 
 +
 
 +
==== 8/24 ====
 +
 
 +
 
 +
'''Transformation'''
 +
 
 +
4-1  (8/23 ligation)
 +
 
 +
17, 18, 19  (8/21 ligation)
 +
 
 +
 
 +
'''Enzyme digestion'''
 +
 
 +
4-4 (EScut,insert)  (8/23 miniprep)
 +
 
 +
12-234 (XPcut,insert)  (8/23 miniprep)
 +
 
 +
14 (SPcut,vector)  (8/17 miniprep)
 +
 
 +
15 (EXcut、vector)  (8/19 miniprep)
 +
 
 +
 
 +
'''cutcheck'''
 +
 
 +
44 ES -> failure & recheck
 +
 
 +
12-234 XP -> failure & recheck
 +
 
 +
14 SP
 +
 
 +
15 EX
 +
 
 +
(8/24 enzyme digestion)
 +
 
 +
 
 +
'''Inoculation'''
 +
 
 +
44, 12-2,3,4
 +
 
 +
 
 +
==== 8/25 ====
 +
 
 +
'''PCR'''
 +
 
 +
hin(rev)
 +
 
 +
PCR program
 +
 
 +
94℃　2min
 +
 
 +
94℃ 15sec
 +
 
 +
64.4℃　30sec
 +
 
 +
68℃ 45sec
 +
 
 +
x25times
 +
 
 +
68℃　7min
 +
 
 +
4℃　forever
 +
 
 +
 
 +
'''Inoculation'''
 +
 
 +
4-1 (8/24 transformation) x2
 +
17, 18, 19
 +
 
 +
 
 +
'''Miniprep'''
 +
 
 +
4-4
 +
 
 +
VII  74.5ng/uL
 +
 
 +
VIII 83.2ng/uL
 +
 
 +
IX  78.4ng/uL
 +
 
 +
X  96.1ng/uL
 +
 
 +
12-234
 +
 
 +
VII  36.7ng/uL
 +
 
 +
VIII  35.3ng/uL
 +
 
 +
IX 43.2ng/uL
 +
 
 +
X 42.4ng/uL
 +
 
 +
5
 +
 
 +
I  91.3ng/uL
 +
 
 +
II  83.9ng/uL
 +
 
 +
4-12
 +
 
 +
I  42.9ng/uL
 +
 
 +
II  38.1ng/uL
 +
 
 +
(8/24 Inoculation)
 +
 
 +
 
 +
'''ligation'''
 +
 
 +
17ES (vector, 4.5uL)
 +
 
 +
18XP (vector, 4.5uL)
 +
 
 +
 
 +
'''Annealing'''
 +
 
 +
rbs rev 289.0ng/uL
 +
 
 +
rbs anti rev 174.9ng/ul
 +
 
 +
location
 +
 
 +
1 281.9ng/uL
 +
 
 +
2 330.6ng/uL
 +
 
 +
3 292.4ng/uL
 +
 
 +
4 235.5ng/uL
 +
 
 +
linker gt EX 267.0ng/uL
 +
 
 +
linker gt SP 291.8ng/uL
 +
 
 +
 
 +
'''lox71 rescue'''
 +
 
 +
iii-lox71
 +
 
 +
PCR program
 +
 
 +
94℃　2min
 +
 
 +
94℃ 15sec
 +
 
 +
64.4℃　30sec
 +
 
 +
68℃ 45sec
 +
 
 +
x25times
 +
 
 +
68℃　7min
 +
 
 +
4℃　forever
 +
 
 +
 
 +
'''Gel extract'''
 +
 
 +
hinrev  8ng/uL
 +
 
 +
 
 +
'''Cutcheck'''
 +
 
 +
4-4(VII~X), 12-234(VII~X)  (8/25 Miniprep)
 +
 
 +
 
 +
==== 8/26 ====
 +
 
 +
'''restriction enzyme digestion'''
 +
 
 +
5-I  EXcut  (8/25 miniprep)
 +
 
 +
 
 +
'''Ligation'''
 +
 
 +
location 1 & gt-EX
 +
 
 +
location 1 & gt-SP 
 +
 
 +
(8/25  annealing)
 +
 
 +
 
 +
'''Miniprep'''
 +
 
 +
4-1 I　71.5ng/uL
 +
 
 +
4-1 II  63.4ng/uL
 +
 
 +
(8/25 Inoculation)
 +
 
 +
 
 +
'''PCR & purification'''
 +
 
 +
MS2 RT
 +
 
 +
PCR program
 +
 
 +
94℃　2min
 +
 
 +
94℃ 15sec
 +
 
 +
63℃　30sec
 +
 
 +
68℃ 4min
 +
 
 +
x25times
 +
 
 +
68℃　1min
 +
 
 +
4℃　forever
 +
 
 +
 
 +
'''transformation'''
 +
 
 +
17, 18  (8/25 Inoculation)
 +
 
 +
 
 +
'''Cutcheck'''
 +
 
 +
4-4 VII 6.5ng/uL
 +
 
 +
4-12 I  5.9ng/uL
 +
 
 +
4-12 II 5.6ng/uL
 +
 
 +
12-234 VII  12.8ng/uL
 +
 
 +
12-234 VIII  8.6ng/uL
 +
 
 +
12-234 IX  8.1ng/uL
 +
 
 +
12-234 X  8.4ng/uL
 +
 
 +
5-I  17.5ng/uL
 +
 
 +
bioA  13.7ng/uL
 +
 
 +
4-12 I  vector  13.2ng/uL
 +
 
 +
4-12 II  vector  12.0ng/uL
 +
 
 +
 
 +
'''sequencing(PCR)'''
 +
 
 +
sudoku（8/4） II
 +
 
 +
PCR program
 +
 
 +
96℃ 1min
 +
 
 +
i = 0
 +
 
 +
while i < 25:
 +
 
 +
98℃ 10sec
 +
 
 +
50℃　5sec
 +
 
 +
60℃　4min
 +
 
 +
i += 1
 +
 
 +
while 1
 +
 
 +
4℃
 +
 
 +
(use 7/20 protocol of sudoku)
 +
 
 +
 
 +
==== 8/27 ====
 +
 
 +
'''hinPCR'''
 +
 
 +
hinrev
 +
 
 +
 
 +
PCR program
 +
 
 +
94℃　2min
 +
 
 +
94℃ 15sec
 +
 
 +
64.4℃　30sec
 +
 
 +
68℃ 45sec
 +
 
 +
x25times
 +
 
 +
68℃　7min
 +
 
 +
4℃ forever
 +
 
 +
 
 +
'''PCR(MS2 RT)'''
 +
 
 +
MS2 RT
 +
 
 +
 
 +
PCR program
 +
 
 +
94℃　2min
 +
 
 +
94℃ 15sec
 +
 
 +
64.4℃　30sec
 +
 
 +
68℃ 4min
 +
 
 +
x25times
 +
 
 +
68℃  1min
 +
 
 +
4℃ forever
 +
 
 +
 
 +
'''Enzyme digestion'''
 +
 
 +
XPcut location  (8/26 ligation)
 +
 
 +
 
 +
'''Gel extract'''
 +
 
 +
hin(rev) & MS2-RT  (8/27 PCR)
 +
 
 +
location  (8/27 XPcut)
 +
 
 +
 
 +
'''PCR'''
 +
 
 +
?????
 +
 
 +
 
 +
==== 8/27 ====
 +
 
 +
'''PCR(retry)'''
 +
 
 +
hinrev
 +
 
 +
 
 +
PCR program
 +
 
 +
94℃　2min
 +
 
 +
94℃ 15sec
 +
 
 +
68℃ 45sec
 +
 
 +
x35times
 +
 
 +
68℃　7min
 +
 
 +
4℃　forever
 +
 
 +
 
 +
'''Transformation'''
 +
 
 +
17, 4-5 & 14-12-234  (8/27 ligation)
 +
 
 +
 
 +
==== 8/29 ====
 +
 
 +
'''Inoculation'''
 +
 
 +
18 III, IV
 +
 
 +
19 III, IV
 +
 
 +
(8/28 transformation)
 +
 
 +
12 III, IV
 +
 
 +
 
 +
'''transformation'''
 +
 
 +
parts1 & sudoku2(rbs)
 +
 
 +
 
 +
'''PCR'''
 +
 
 +
MS2(EX-SP)
 +
 
 +
 
 +
PCRprogram
 +
 
 +
94℃ 2min
 +
 
 +
94℃ 15sec
 +
 
 +
60℃ 30sec
 +
 
 +
68℃ 4min30sec
 +
 
 +
x30
 +
 
 +
68℃ 60sec
 +
 
 +
4℃ forever
 +
 
 +
 
 +
'''Ligation'''
 +
 
 +
17 EScut  (8/21 annealing)
 +
 
 +
4-I EScut (insert) & 5-I EXcut (vector)
 +
 
 +
12-234VII XPcut  (8/26 gel extract) & 14 SPcut (8/24 gel extract)
 +
 
 +
 
 +
'''Enzyme digestion'''
 +
 
 +
XPcut 234  (7/6 miniprep)
 +
 
 +
EScut 4-II  (8/17 miniprep)
 +
 
 +
SPcut 12-I  (8/4 miniprep)
 +
 
 +
 
 +
'''Gel extract'''
 +
 
 +
PCR product of MS2 PCR
 +
 
 +
3600bp 7.0ng/uL
 +
 
 +
2000bp 6.ong/uL
 +
 
 +
 
 +
==== 8/30 ====
 +
 
 +
'''miniprep'''
 +
 
 +
18-I  56.5ng/uL
 +
 
 +
18-II  71.8ng/uL
 +
 
 +
18-III  45.5ng/uL
 +
 
 +
18-IV  85.6ng/uL
 +
 
 +
19-I  52.3ng/uL
 +
 
 +
19-II  44.9ng/uL
 +
 
 +
19-III  43.5ng/uL
 +
 
 +
19-IV  50.7ng/uL
 +
 
 +
12-III  54.9ng/uL
 +
 
 +
 
 +
'''Gel extract'''
 +
 
 +
234  XPcut
 +
 
 +
4-Ⅱ  EScut
 +
 
 +
12-I  SPcut
 +
 
 +
PCR product of MS2
 +
 
 +
 
 +
'''Ligation'''
 +
 
 +
lox71  (8/25)
 +
 
 +
12-I SP  8.3ng/uL  (vector, 8/30 gel extract)
 +
 
 +
4-12 XP  5.6ng/uL  (insurt, 8/26 gel extract)
 +
 
 +
 
 +
'''Enzyme digestion'''
 +
 
 +
EXcut 18 II, IV  (8/30 miniprep)
 +
 
 +
 
 +
'''Phosphorylation'''
 +
 
 +
location 1~4, rbs, rbs anti
 +
 
 +
 
 +
'''Ligation'''
 +
 
 +
location 1 EXcut
 +
 
 +
location 1 SPcut
 +
 
 +
(8/27 gel extract)
 +
 
 +
 
 +
'''Cut check'''
 +
 
 +
MS2 PCR product
 +
 
 +
 
 +
PCRprogram
 +
 
 +
94℃　2min
 +
 
 +
94℃ 15sec
 +
 
 +
68℃ 4min
 +
 
 +
68℃ 60sec
 +
 
 +
x35times
 +
 
 +
4℃ forever
 +
 
 +
 
 +
==== 8/31 ====
 +
 
 +
'''Inoculation'''
 +
 
 +
lox71-rescue, flpe1st,
 +
 
 +
empty vector, 12-4-12
 +
 
 +
 
 +
'''Ligation'''
 +
 
 +
location2, 3, 4 (8/30 Phosphorylation)
 +
 
 +
 
 +
 
 +
{{UT-Tokyo_Foot}}

Latest revision as of 03:02, 28 October 2010

UT-Tokyo

Sudoku


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


Note Book

8/4

Inoculation

sudoku 12-I & 12-II

sudoku 13-I & 13-II

(8/3 Transformation)


Enzyme digestion

EScut i 1-99234I (7/24 Miniprep & Sequencing)

EScut ii 1-9234I (7/21 Miniprep / 7/24 Sequencing)

EXcut iii 1,5,11,7,8,4II (7/29 Miniprep)　


Gel extract

i 1-99234 8.8ng/uL

ii 1-9234 7.1ng/uL

iii 1-5-11-7-8-4 18.0ng/uL


Ligation

i-iii, ii-iii

(8/4 Miniprep)


Miniprep

12-i　58.7ng/uL

12-ii　55.9ng/uL

13-i　48.7ng/uL

13-ii　48.5ng/uL

(8/4 Inoculation)


8/5

Enzyme digestion

1234 107.2ng/uL (8/4 gel extract)


Gel extract

sudoku (1234) 6.6ng/uL


Ligation

0(1234)&iii


Transformation

i-iii & ii-iii

0(1234)-iii

(8/4 & 8/5)


8/6

Inoculation

0-iii, (i-iii), ii-iii (8/5 transformation)


transformation

i-iii (8/4 ligation)


8/9

Enzyme digestion

0 107.2ng/uL (7/10 Miniprep)

iii 100.7ng/uL (7/29 Miniprep ii)


Gel extract

0 & iii 56.6ng/uL

(8/9 Enzyme digestion)


Sequencing

iii-II (7/29 Miniprep)


8/10

Cutcheck

XScut 0-iii, I, II (8/9 ligation)

-> failure


transformation

0-iii (8/9 ligation)


8/11

Inoculation

sudoku 0-iii I,II,III,IV

(8/10 transformation)


Miniprep

I 21.2ng/uL

II 18.8ng/uL

III 70.6ng/uL

IV 122.3ng/uL

(8/11 Inoculation)


Cutcheck

I, II, III, IV


8/12

Transformation

sudoku iii-II(7/29)


8/13

colonyPCR

sudoku 0-iii (x16)

program PCR

94℃ 10min

{94℃ 20sec 53℃ 20sec 72℃ 3min}x35

4℃ forever


Sequencing(ethanol precipitation)

master mix(8/13)


8/14

Miniprep

iii(II)-Ⅰ 113.4 ng/uL

iii(II)-II 98.9 ng/uL

iii(II)-III 153.2 ng/uL

(8/13 Inoculation)


8/16

Enzyme digestion

EXcut 1,12

EScut 4

SPcut 12

XPcut 2,3,4


Gel extract

1,2,3,4,12


ligation

12-2,3,4


Inoculation

4,14


transformation

12-2,3,4


8/18

Gel extract

4-1 EXcut 20.6ng/uL

4-2 EScut 4.3ng/uL

(8/17 enzyme digestion)


Inoculation

15 (8/17 transformation)


8/19

Miniprep

15-I, 15-II (8/18 Inoculation)

15-I　75.3ng/uL

15-II　74.8ng/uL


Ligation

12 EXcut 21.6ng/uL (8/16 enzyme digestion & gel extract)

4-I EScut 4.3ng/uL (8/17 enzyme digestion & 8/18 gel extract)

4-I EXcut  ?ng/uL (8/17 enzyme digestion & 8/18 gel extract)

4-12

vector 12EX　7.7ul

insert 4ES 2.3ul

4-4

vector 4EX 8uL

insert 4ES 2uL


Transformation

4-1, 4-12, 4-4 (8/19 ligation)

12-234 (8/16 ligation)


Enzyme digestion

SPcut 12-I (8/4 Miniprep)


8/20

Enzyme digestion

EXcut 12-I (8/4 Miniprep)


Ligation

12-I SPcut 10.2ng/uL, 2000bp (8/20 gel extract) &

2,3,4 XPcut 10.2ng/uL, 1100bp (7/8 gel extract)

12-I SPcut 10.2ng/uL (8/20 gel extract) &

2,3,4 XPcut 9.1ng/uL (8/16 enzyme digestion & gel extract)


Transformation

12-2,3,4 (8/20 ligation) x2


8/21

Miniprep

4-4 (I, II) (8/20 inoculation)


Annealing

PSP6revES（17） f & r 232.2ng/uL

Lox2272XP（18） f & r 476.3ng/uL

loadingrevXP（19） f & r 363.8ng/uL


Inoculation

sudoku1 & 4-4


Ligation

pSP6revES(17) 1uL (vector)

lox2272XP(18) 1uL (vector)

loading revXP(19) 1uL (vector)

4-I EScut 4.3ng/uL, 95bp (8/19 enzyme digestion)

12 EXcut 21.6ng/uL, 2000bp (8/16 enzyme digestion)


Transformation

4-1 (8/19 ligation)

4-12 (8/21 ligation)


Gel extant

12-I (8/19 enzyme digestion)


RT-PCR

cDNA of MS2

PCR program

1. 94℃, 2 minutes

2. 94℃, 15 seconds

3. 55℃, 30 seconds

4. 68℃, 3minutes 3 seconds

5. 2,3,4 x 24

6. 68℃, 4 minutes

7. 4℃, forever


8/22

purification

cDNA of MS2 (8/21 PCR)


Miniprep

1(II)

I　99.2ng/uL

II 112.8ng/uL

(8/21 Inoculation)

4-4

III　45.6ng/uL

IV　33.4ng/uL

(8/21 Inoculation)

12-2,3,4

I　17.9ng/uL

II　3.3ng/uL

(7/8 ligation & transformation / 8/21 Inoculation)

III　1.1ng/uL

IV　7.3ng/uL

(8/16 ligation & transformation / 8/21 Inoculation)


Inoculation

4-4 & 12-234

(8/22 Miniprep)


XPcut(RT-PCR)

XPcut cDNA of MS2 with Restriction Enzyme sites

(8/22 purification)


Gel extract

12-I EXcut 12.3ng/uL (8/20 enzyme digestion)


Transformation

17, 18, 19 (8/21 ligation)


Enzyme digestion

1(II)-II EXcut (8/22 miniprep)


Ligation

12-I EXcut (8/22 gel extract)

4-I EScut (8/18 gel extract)


8/24

Transformation

4-1 (8/23 ligation)

17, 18, 19 (8/21 ligation)


Enzyme digestion

4-4 (EScut,insert) (8/23 miniprep)

12-234 (XPcut,insert) (8/23 miniprep)

14 (SPcut,vector) (8/17 miniprep)

15 (EXcut、vector) (8/19 miniprep)


cutcheck

44 ES -> failure & recheck

12-234 XP -> failure & recheck

14 SP

15 EX

(8/24 enzyme digestion)


Inoculation

44, 12-2,3,4


8/25

PCR

hin(rev)

PCR program

94℃　2min

94℃ 15sec

64.4℃　30sec

68℃ 45sec

x25times

68℃　7min

4℃　forever


Inoculation

4-1 (8/24 transformation) x2 17, 18, 19


Miniprep

4-4

VII 74.5ng/uL

VIII 83.2ng/uL

IX 78.4ng/uL

X 96.1ng/uL

12-234

VII 36.7ng/uL

VIII 35.3ng/uL

IX 43.2ng/uL

X 42.4ng/uL

5

I 91.3ng/uL

II 83.9ng/uL

4-12

I 42.9ng/uL

II 38.1ng/uL

(8/24 Inoculation)


ligation

17ES (vector, 4.5uL)

18XP (vector, 4.5uL)


Annealing

rbs rev 289.0ng/uL

rbs anti rev 174.9ng/ul

location

1 281.9ng/uL

2 330.6ng/uL

3 292.4ng/uL

4 235.5ng/uL

linker gt EX 267.0ng/uL

linker gt SP 291.8ng/uL


lox71 rescue

iii-lox71

PCR program

94℃　2min

94℃ 15sec

64.4℃　30sec

68℃ 45sec

x25times

68℃　7min

4℃　forever


Gel extract

hinrev 8ng/uL


Cutcheck

4-4(VII~X), 12-234(VII~X) (8/25 Miniprep)


8/26

restriction enzyme digestion

5-I EXcut (8/25 miniprep)


Ligation

location 1 & gt-EX

location 1 & gt-SP

(8/25 annealing)


Miniprep

4-1 I　71.5ng/uL

4-1 II 63.4ng/uL

(8/25 Inoculation)


PCR & purification

MS2 RT

PCR program

94℃　2min

94℃ 15sec

63℃　30sec

68℃ 4min

x25times

68℃　1min

4℃　forever


transformation

17, 18 (8/25 Inoculation)


Cutcheck

4-4 VII 6.5ng/uL

4-12 I 5.9ng/uL

4-12 II 5.6ng/uL

12-234 VII 12.8ng/uL

12-234 VIII 8.6ng/uL

12-234 IX 8.1ng/uL

12-234 X 8.4ng/uL

5-I 17.5ng/uL

bioA 13.7ng/uL

4-12 I vector 13.2ng/uL

4-12 II vector 12.0ng/uL


sequencing(PCR)

sudoku（8/4） II

PCR program

96℃ 1min

i = 0

while i < 25:

98℃ 10sec

50℃　5sec

60℃　4min

i += 1

while 1

4℃

(use 7/20 protocol of sudoku)


8/27

hinPCR

hinrev


PCR program

94℃　2min

94℃ 15sec

64.4℃　30sec

68℃ 45sec

x25times

68℃　7min

4℃ forever


PCR(MS2 RT)

MS2 RT


PCR program

94℃　2min

94℃ 15sec

64.4℃　30sec

68℃ 4min

x25times

68℃ 1min

4℃ forever


Enzyme digestion

XPcut location (8/26 ligation)


Gel extract

hin(rev) & MS2-RT (8/27 PCR)

location (8/27 XPcut)


PCR

?????


8/27

PCR(retry)

hinrev


PCR program

94℃　2min

94℃ 15sec

68℃ 45sec

x35times

68℃　7min

4℃　forever


Transformation

17, 4-5 & 14-12-234 (8/27 ligation)


8/29

Inoculation

18 III, IV

19 III, IV

(8/28 transformation)

12 III, IV


transformation

parts1 & sudoku2(rbs)


PCR

MS2(EX-SP)


PCRprogram

94℃ 2min

94℃ 15sec

60℃ 30sec

68℃ 4min30sec

x30

68℃ 60sec

4℃ forever


Ligation

17 EScut (8/21 annealing)

4-I EScut (insert) & 5-I EXcut (vector)

12-234VII XPcut (8/26 gel extract) & 14 SPcut (8/24 gel extract)


Enzyme digestion

XPcut 234 (7/6 miniprep)

EScut 4-II (8/17 miniprep)

SPcut 12-I (8/4 miniprep)


Gel extract

PCR product of MS2 PCR

3600bp 7.0ng/uL

2000bp 6.ong/uL


8/30

miniprep

18-I 56.5ng/uL

18-II 71.8ng/uL

18-III 45.5ng/uL

18-IV 85.6ng/uL

19-I 52.3ng/uL

19-II 44.9ng/uL

19-III 43.5ng/uL

19-IV 50.7ng/uL

12-III 54.9ng/uL


Gel extract

234 XPcut

4-Ⅱ EScut

12-I SPcut

PCR product of MS2


Ligation

lox71 (8/25)

12-I SP 8.3ng/uL (vector, 8/30 gel extract)

4-12 XP 5.6ng/uL (insurt, 8/26 gel extract)


Enzyme digestion

EXcut 18 II, IV (8/30 miniprep)


Phosphorylation

location 1~4, rbs, rbs anti


Ligation

location 1 EXcut

location 1 SPcut

(8/27 gel extract)


Cut check

MS2 PCR product


PCRprogram

94℃　2min

94℃ 15sec

68℃ 4min

68℃ 60sec

x35times

4℃ forever


8/31

Inoculation

lox71-rescue, flpe1st,

empty vector, 12-4-12


Ligation

location2, 3, 4 (8/30 Phosphorylation)


Retrieved from "http://2010.igem.org/Team:UT-Tokyo/Sudoku_notebook_2"