Sudoku

Abstract Construct Experiment Notebook Result

Notebook

Pre-experiment

AUGUST!!!!!

AUGUST!!!!!

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

AUGUST!!!!!

Make construct

8/2

Sequencing

sequel to 7/31

add HiDi 20μL

A6 I pre, B6 I sur,C6 II pre

D6 II sur, E6 III pre, F6 III sur

8/4

Miniprep

12－Ⅰ　58.7ng/uL

12－Ⅱ　55.9ng/uL

13ーⅠ　48.7ng/uL

13ーⅡ　48.5ng/uL

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)　

Ligation

i-iii i:6ul iii:6ul ligationmix 12ul

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

Gel extract

i 1-99234 8.8ng/uL

ii 1-9234 7.1ng/uL

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

8/5

Enzyme digestion

1234（107.2ng/uL）

Gel extract

sudoku (1234) 6.6ng/ul

Ligation

0(1234)&iii

7/5

Ligation

2-3 (7/3 enzyme cut) & 4 / 11 (7/3 enzyme cut) & 7

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)

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)