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

i 1-99234Ⅰ EScut (7/24 Miniprep & Sequencing)

ii 1-9234Ⅰ EScut (7/21 Miniprep / 7/24 Sequencing)

iii 1,5,11,7,8,4Ⅱ EXcut (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 i:6uL iii:6uL ligationmix 12uL

ii-iii ii:6uL iii:4uL ligationmix 10uL

(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)

Gel extract

0-iii I 20uL

0-iii II 20uL

0-iii III 20uL

0-iii IV 20uL

SEG 1 XPcut 20uL

SEG 1 EScut 20uL

Ligation

linker EXS +Omp A

linker EXP +Cryj1

linker EXP + Cryj2

8/13

colonyPCR

sudoku 0-iii (x16)

program PCR

94℃ 10min

{94℃ 20sec 53℃ 20sec 72℃ 3min}×35

4℃ forever

Sequencing(ethanol precipitation)

master mix(8/13)

8/14

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)