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

8/12

8/12

8/12

8/12

8/12

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)