Sudoku

Abstract Construct Experiment Notebook Result

Notebook

Pre-experiment

AUGUST!!!!!

AUGUST!!!!!

AUGUST!!!!! AUGUST!!!!!

AUGUST!!!!!

Make construct

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, 19 (8/28 transformation)

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)

Inoculation

18 III, IV

19 III, IV

12 III, IV

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

8/31

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)