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Team:Tokyo Metropolitan/Notebook/S16
From 2011.igem.org
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@electrophoresis for confirmation, but could not find bind. | @electrophoresis for confirmation, but could not find bind. | ||
| + | |||
| + | |||
| + | ---------------------------------------------------------------------------- | ||
| + | -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 | ||
| - | + | 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 |
| - | + | 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 | + | 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 | could not find both of the sequence | ||
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@Visual | @Visual | ||
| - | + | 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 | ||
| - | + | no.6 OriT_GFP_OriT_1 / GFP | |
| - | + | no.7 OriT_GFP_OriT_1 / VR | |
| - | could read about 650 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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| - | + | 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 | ||
| - | + | 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 | ||
| - | + | 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 | ||
| - | + | 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 | ||
| - | + | no.12 OriT_GFP_OriT_3 / GFP | |
could not read sequence | could not read sequence | ||
| - | + | 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 | ||
| - | + | 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 | ||
| - | + | 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 | ||
| - | + | 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 | |
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 | |
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. | ||
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| + | = -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
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-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
