Team:Tokyo Metropolitan/Notebook/S16

From 2011.igem.org

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(-Visualization-)
 
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@electrophoresis for confirmation, but could not find bind.
@electrophoresis for confirmation, but could not find bind.
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 +
 +
----------------------------------------------------------------------------
 +
-Digestion
 +
 +
Promoter: Spe and Pst
 +
 +
Suppressed expression: Xba and Pst
 +
 +
 +
-Electrophoresis
 +
 +
Above samples
 +
 +
 +
-Gel extraction
 +
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Above samples
 +
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@Killer+Target
@Killer+Target
-
①J23100-K317037-K283030 / VF
+
no.1J23100-K317037-K283030 / VF
-
could read 500 base which contain vector and the first half of J23100 and K317037, but not bond to
+
could read 500 base which contain vector and the first half of J23100 and K317037, but not bond to no.2
-
②J23100-K317037-K283030 / VR
+
no.2 J23100-K317037-K283030 / VR
could read 500 base which contain vector and the latter half of K283030,
could read 500 base which contain vector and the latter half of K283030,
-
but could not bond to , deficiene 50 base from terminator codon of CheZ in K283030 (also terminator codon).
+
but could not bond to no.1, deficiene 50 base from terminator codon of CheZ in K283030 (also terminator codon).
-
③P0140 / VF
+
no.3 P0140 / VF
-
④P0140 / VR
+
no.4 P0140 / VR
could not find both of the sequence
could not find both of the sequence
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@Visual
@Visual
-
⑤OriT_GFP_OriT_1 / VF
+
no.5 OriT_GFP_OriT_1 / VF
could read about 350 base and confirmate vector and part of OriT for 60 base
could read about 350 base and confirmate vector and part of OriT for 60 base
-
⑥OriT_GFP_OriT_1 / GFP
+
no.6 OriT_GFP_OriT_1 / GFP
-
⑦OriT_GFP_OriT_1 / VR
+
no.7 OriT_GFP_OriT_1 / VR
-
could read about 650 base from and about 350 base from ,
+
could read about 650 base from no.6 and about 350 base from no.7,
and bond each sequence to one
and bond each sequence to one
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-
⑧OriT_GFP_OriT_2 / VF
+
no.8 OriT_GFP_OriT_2 / VF
could read about 350 base and confirmate vector and part of OriT for 60 base
could read about 350 base and confirmate vector and part of OriT for 60 base
-
⑨OriT_GFP_OriT_2 / GFP
+
no.9 OriT_GFP_OriT_2 / GFP
could read about 600 base, but that is just sequence of GFP
could read about 600 base, but that is just sequence of GFP
-
⑩OriT_GFP_OriT_2 / VR
+
no.10 OriT_GFP_OriT_2 / VR
could read 300 base, but not agree with OriT_GFP_OriT
could read 300 base, but not agree with OriT_GFP_OriT
-
⑪OriT_GFP_OriT_3 / VF
+
no.11 OriT_GFP_OriT_3 / VF
could read 250 base which contain OriT for 60 base and vector for other sequence
could read 250 base which contain OriT for 60 base and vector for other sequence
-
⑫OriT_GFP_OriT_3 / GFP
+
no.12 OriT_GFP_OriT_3 / GFP
could not read sequence
could not read sequence
-
⑬OriT_GFP_OriT_3 / VR
+
no.13 OriT_GFP_OriT_3 / VR
could read about 300 base and find sequence of OriT
could read about 300 base and find sequence of OriT
-
⑭OriT_GFP_OriT_4 / VF
+
no.14 OriT_GFP_OriT_4 / VF
could read 200 base and confirmated vector and the first half of OriT
could read 200 base and confirmated vector and the first half of OriT
-
⑮OriT_GFP_OriT_4 / GFP
+
no.15 OriT_GFP_OriT_4 / GFP
could read about 600 base, but that is just sequence of GFP
could read about 600 base, but that is just sequence of GFP
-
⑯OriT_GFP_OriT_4 / VR
+
no.16 OriT_GFP_OriT_4 / VR
could read about 300 base and confirmated the latter half of OriT
could read about 300 base and confirmated the latter half of OriT
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@summary
@summary
-
Targeting
+
Targeting
succeed in ligation, but deficiene 50 base from terminator codon of CheZ in K28303.
succeed in ligation, but deficiene 50 base from terminator codon of CheZ in K28303.
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-
Visual
+
Visual
4 sumples of OriT-GFP-OriT was turned out wrong.
4 sumples of OriT-GFP-OriT was turned out wrong.
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3 was OriT only, which is self ligation.
3 was OriT only, which is self ligation.
 +
 +
 +
= -Targetting- =
 +
 +
@preculture P0140, J23100-K317037-K283030(Targetting Device), pSB1C3
 +
 +
@plasmid extraction and DNA refine P0140, J23100-K317037-K283030(Targeting Device), pSB1C3
 +
 +
@restriction enzyme(XbaⅠ, Pst) P0140, J23100-K317037-K283030(Targeting Device), pSB1C3

Latest revision as of 13:34, 5 October 2011

-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.



-Digestion

Promoter: Spe and Pst

Suppressed expression: Xba and Pst


-Electrophoresis

Above samples


-Gel extraction

Above samples


-Sequencing-

@Killer+Target

no.1J23100-K317037-K283030 / VF

could read 500 base which contain vector and the first half of J23100 and K317037, but not bond to no.2


no.2 J23100-K317037-K283030 / VR

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

but could not bond to no.1, deficiene 50 base from terminator codon of CheZ in K283030 (also terminator codon).


no.3 P0140 / VF

no.4 P0140 / VR

could not find both of the sequence


@Visual

no.5 OriT_GFP_OriT_1 / VF

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


no.6 OriT_GFP_OriT_1 / GFP

no.7 OriT_GFP_OriT_1 / VR

could read about 650 base from no.6 and about 350 base from no.7,

and bond each sequence to one


no.8 OriT_GFP_OriT_2 / VF

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


no.9 OriT_GFP_OriT_2 / GFP

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


no.10 OriT_GFP_OriT_2 / VR

could read 300 base, but not agree with OriT_GFP_OriT


no.11 OriT_GFP_OriT_3 / VF

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


no.12 OriT_GFP_OriT_3 / GFP

could not read sequence


no.13 OriT_GFP_OriT_3 / VR

could read about 300 base and find sequence of OriT


no.14 OriT_GFP_OriT_4 / VF

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


no.15 OriT_GFP_OriT_4 / GFP

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


no.16 OriT_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.


-Targetting-

@preculture P0140, J23100-K317037-K283030(Targetting Device), pSB1C3

@plasmid extraction and DNA refine P0140, J23100-K317037-K283030(Targeting Device), pSB1C3

@restriction enzyme(XbaⅠ, Pst) P0140, J23100-K317037-K283030(Targeting Device), pSB1C3