-Visualization-

@plasmid extraction oriTr-GFP-oriTr and JM109

@plasmid extraction and DNA refine oriTr-GFP-oriTr and JM109

@DNA densitometry

oriTr-GFP-oriTr 5 26.1 ng/μl

oriTr-GFP-oriTr 6 51.3 ng/μl

oriTr-GFP-oriTr 7 47.2 ng/μl

oriTr-GFP-oriTr 8 58.4 ng/μl

@restriction enzyme to oriTr-GFP-oriTr (XbaⅠ, PstⅠ)

@electrophoresis and gel extraction oriTr-GFP-oriTr5～8 and pSB1C3(restriction enzymed 9/15)

@DNA densitometry

totaled oriTr-GFP-oriTr5,6,8 18.4 ng/μl

pSB1C3① 16.5 ng/μl

pSB1C3② 11.5 ng/μl

@electrophoresis for confirmation, but could not find bind.

-Sequencing-

@Killer＋Target

①J23100-K317037-K283030　/　VF

could read 500 base which contain vector and the first half of J23100 and K317037, but not bond to ②

②J23100-K317037-K283030　/　VR

could read 500 base which contain vector and the latter half of K283030,

but could not bond to ①, deficiene 50 base from terminator codon of CheZ in K283030 (also terminator codon).

③P0140　/　VF

④P0140　/　VR

could not find both of the sequence

@Visual

⑤OriT_GFP_OriT_1　/　VF

could read about 350 base and confirmate vector and part of OriT for 60 base

⑥OriT_GFP_OriT_1　/　GFP

⑦OriT_GFP_OriT_1　/　VR

could read about 650 base from ⑥ and about 350 base from ⑦,

and bond each sequence to one

⑧OriT_GFP_OriT_2　/　VF

could read about 350 base and confirmate vector and part of OriT for 60 base

⑨OriT_GFP_OriT_2　/　GFP

could read about 600 base, but that is just sequence of GFP

⑩OriT_GFP_OriT_2　/　VR

could read 300 base, but not agree with OriT_GFP_OriT

⑪OriT_GFP_OriT_3　/　VF

could read 250 base which contain OriT for 60 base and vector for other sequence

⑫OriT_GFP_OriT_3　/　GFP

could not read sequence

⑬OriT_GFP_OriT_3　/　VR

could read about 300 base and find sequence of OriT

⑭OriT_GFP_OriT_4　/　VF

could read 200 base and confirmated vector and the first half of OriT

⑮OriT_GFP_OriT_4　/　GFP

could read about 600 base, but that is just sequence of GFP

⑯OriT_GFP_OriT_4　/　VR

could read about 300 base and confirmated the latter half of OriT

@summary

Targeting

succeed in ligation, but deficiene 50 base from terminator codon of CheZ in K28303.

I guessed there is not His-tag.

could not read sequence of P0140.

decrease tenpate concentration and sequencing again

Visual

4 sumples of OriT-GFP-OriT was turned out wrong.

1 and 4 were OriT-GFP.

2 was unknown.

3 was OriT only, which is self ligation.