Team:Kyoto/Notebook

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{{:Team:Kyoto/Header}}
{{:Team:Kyoto/Header}}
-
==Index==
 
==Notebook==
==Notebook==
-
===Tuesday, July 20 <span class="by">By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto</span>===
+
===Notebooks===
-
<div class="transformation lysis-construction measure-construction">
+
* [[Team:Kyoto/Notebook1|Notebook1]]: Construction for Lysisbox.
-
====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
+
* [[Team:Kyoto/Notebook2|Notebook2]]: Measurement of R0011.
-
{| class="experiments"
+
* [[Team:Kyoto/Notebook3|Notebook3]]: Measurement of Lysis Cassette and Lysisbox etc.
-
!Name||Well||Sample (&micro;l)||Competent Cells (&micro;l)||Total (&micro;l)||Plate||Incubation||Result
+
-
|-
+
-
|<partinfo>J23100</partinfo>||1-18-C||1||20||21||rowspan="7"|LB (Amp+)||rowspan="8"|At 37&#x2103;, 7/20 20:50 - 7/21 17:00||&#x25CB;
+
-
|-
+
-
|<partinfo>J23105</partinfo>||1-18-M||1||20||21||&#x25CB;
+
-
|-
+
-
|<partinfo>J23116</partinfo>||1-20-M||1||20||21||&#x25CB;
+
-
|-
+
-
|<partinfo>R0011</partinfo>||1-6-G||1||20||21||&#x25CB;
+
-
|-
+
-
|<partinfo>E0840</partinfo>||1-12-O||1||20||21||&#x25CB;
+
-
|-
+
-
|<partinfo>J06702</partinfo>||2-8-E||1||20||21||&#x25CB;
+
-
|-
+
-
|<partinfo>pSB4K5</partinfo>||1-5-G||1||20||21||&#xD7;
+
-
|-
+
-
|<partinfo>B0015</partinfo>||1-23-L||1||20||21||LB (Kan+)||&#xD7;
+
-
|}
+
-
A vector of <partinfo>pSB4K5</partinfo> is Kanamycin-resistance, however, we plated it to LB plate (Amp+). And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance. So, it was predicted that we will fail the transformation of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo>.
+
-
</div>
+
-
===Wednesday, July 21 <span class="by">By: Wataru, Ken, Makoto, Takuya Y.</span>===
+
[[#top-section|^Top]]
-
<div class="culture lysis measure">
+
-
====Culture of plates in which colonies was observed at 37&#x2103; from 07/21 20:50 to 07/22 17:00====
+
-
</div>
+
-
<div class="master lysis measure">
+
===Other Information===
-
====Making a master plate of the above plates====
+
* [[Team:Kyoto/Protocols|Protocols]]: Protocols of each experiment such as Polymerase Chain Reaction (PCR), Restriction Digestion, Ligation, Transformation.
-
</div>
+
* [[Team:Kyoto/Materials|Materials]]: Strains, DNA, and Primers.
 +
* [[Team:Kyoto/Parts|Parts]]: Construction of each part and BioBrick Parts used in our project.
-
<div class="transformation lysis measure">
+
[[#top-section|^Top]]
-
====Retry [[Team:Kyoto/Protocols#Transformation|Transformation]]====
+
-
{| class="experiments"
+
-
!Name||Well||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
+
-
|-
+
-
|<partinfo>pSB4K5</partinfo>||1-5-G||1||20||21||rowspan="2"|LB (Kan+)||rowspan="2"|At 37℃ 7/21 20:50 - 7/22 16:30||○
+
-
|-
+
-
|<partinfo>B0015</partinfo>||1-23-L||1||20||21||○
+
-
|}
+
-
</div>
+
-
<div class="pcr lysis">
 
-
====[[Team:Kyoto/Protocols#Stantard_PCR|PCR]] for S-R-Rz/Rz1 and S====
 
-
{| class="experiments"
 
-
!No.||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||TemplateDNA (5ng/µl)||Primer Forward (10µM)||Primer S-R-Rz/Rz1 Reverse (10µM)||Primer S Reverse (10µM)||KOD Plus ver.2||Total
 
-
|-
 
-
|1||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 
-
|-
 
-
|2||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 
-
|-
 
-
|3||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 
-
|-
 
-
|4||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 
-
|-
 
-
|5||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 
-
|-
 
-
|6||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 
-
|-
 
-
|7||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 
-
|-
 
-
|8||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 
-
|}
 
-
{|class="experiments"
 
-
|-
 
-
|94&#x2103;||2min||
 
-
|-
 
-
|98&#x2103;||10sec||rowspan="3"|30 cycles
 
-
|-
 
-
|55&#x2103;||30sec
 
-
|-
 
-
|68&#x2103;||4min
 
-
|-
 
-
|4&#x2103;||forever||
 
-
|}
 
-
</div>
 
-
 
-
 
-
===Thursday, July 22 <span class="by">By: Wataru</span>===
 
-
<div class="electrophoresis lysis">
 
-
====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] of the PCR products for 40min====
 
-
[[Image:KyotoExp100722-1.png|300px|right]]
 
-
Length of S and S-R-Rz/Rz1 is 370bp and 1300bp, so PCR succeeded.
 
-
</div>
 
-
 
-
<div class="miniprep lysis measure">
 
-
====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
 
-
{| class="experiments"
 
-
!Name||Concentration(ng/&micro;l)
 
-
|-
 
-
|<partinfo>J23100</partinfo>||18.5
 
-
|-
 
-
|<partinfo>J23105</partinfo>||12.5
 
-
|-
 
-
|<partinfo>J23116</partinfo>||14.6
 
-
|-
 
-
|<partinfo>R0011</partinfo>||8.6
 
-
|-
 
-
|<partinfo>E0840</partinfo>||12.1
 
-
|-
 
-
|<partinfo>J06702</partinfo>||14.7
 
-
|}
 
-
The concentration of all samples was very week. Probably our shaking incubation was week.
 
-
</div>
 
-
 
-
<div class="culture lysis">
 
-
====Culture of plates and making master plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo> from 07/22 17:00 to 07/23 10:00====
 
-
</div>
 
-
 
-
 
-
===Friday, July 23 <span class="by">By: Wataru, Tomo, Makoto</span>===
 
-
<div class="miniprep lysis">
 
-
====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
 
-
{| class="experiments"
 
-
!Name||Concentration(ng/&micro;l)
 
-
|-
 
-
|<partinfo>pSB4K5</partinfo>||79.2
 
-
|-
 
-
|<partinfo>B0015</partinfo>||-
 
-
|}
 
-
We lost <partinfo>B0015</partinfo> by our mistake. The concentration of <partinfo>pSB4K5</partinfo> is high, so this condition of shaking incubation is moderate.
 
-
</div>
 
-
 
-
<div class="pcr-purification lysis">
 
-
====Picking up number 1, 3, 5, and 7 of the products of PCR, and PCR-purification====
 
-
{| class="experiments"
 
-
!No.||Name||Concentration (ng/&micro;l)||New Name
 
-
|-
 
-
|1||S-R-Rz/Rz1||18.6||-
 
-
|-
 
-
|3||S||77.6||S<sub>1</sub>
 
-
|-
 
-
|5||S-R-Rz/Rz1||33.6||-
 
-
|-
 
-
|7||S||65.4||S<sub>2</sub>
 
-
|}
 
-
The concentration of sample number 1 and 5, the PCR products of S-R-Rz/Rz1, is week, so we desided to retry PCR.
 
-
</div>
 
-
 
-
<div class="pcr lysis">
 
-
====Retry of [[Team:Kyoto/Protocols#Standard_PCR|Standard PCR]] for S-R-Rz/Rz1====
 
-
{| class="experiments"
 
-
!No.||Water||25mmol/l MgSO4||2mmol/l dNTPs||10×Buffer for KOD plus ver.2||Template DNA (5ng/&micro;l)||Primer S-R-Rz/Rz1 Forward (10&micro;mol/l)||Primer S-R-Rz/Rz1 Reverse (10&micro;mol/l)||KOD plus ver.2||Total
 
-
|-
 
-
|1||28&micro;l||3||5||5||5||1.5||1.5||1||50
 
-
|-
 
-
|2||28||3||5||5||5||1.5||1.5||1||50
 
-
|-
 
-
|3||26.5||4.5||5||5||5||1.5||1.5||1||50
 
-
|-
 
-
|4||26.5||4.5||5||5||5||1.5||1.5||1||50
 
-
|-
 
-
|5||25||6||5||5||5||1.5||1.5||1||50
 
-
|-
 
-
|6||25||6||5||5||5||1.5||1.5||1||50
 
-
|}
 
-
{|class="experiments"
 
-
|+ PCR condition
 
-
|-
 
-
|94&#x2103;||2min||
 
-
|-
 
-
|98&#x2103;||10sec||rowspan="3"|30 cycles
 
-
|-
 
-
|55&#x2103;||30sec
 
-
|-
 
-
|68&#x2103;||4min
 
-
|-
 
-
|4&#x2103;||forever||
 
-
|}
 
-
</div>
 
-
 
-
<div class="digestion">
 
-
====[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]] of <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, and PstI to check function of our Restriction Enzyme====
 
-
{| class="experiments"
 
-
!No.||10xBuffer||BSA||Enzyme||MilliQ||Total||Incubation
 
-
|-
 
-
|1||5&micro;l||1||''EcoR''I 0.1||3.6||10||rowspan="5"|At 37&#x2103; 7/23 18:00 - 7/23 18:30
 
-
|-
 
-
|2||5||1||''Xba''I 0.1||3.6||10
 
-
|-
 
-
|3||5||1||''Spe''I 0.1||3.6||10
 
-
|-
 
-
|4||5||1||''Pst''I 0.1||3.6||10
 
-
|-
 
-
|5||5||1||-||3.7||10
 
-
|}
 
-
</div>
 
-
 
-
<div class="electrophoresis">
 
-
====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] of above sample for 35min====
 
-
[[Image:KyotoExp100723-1.png|300px|right]]
 
-
Comparison to sample 5(control, circular DNA), the bands of sample 1, 2, 3, 4 was shifted. The DNA of sample 1, 2, 3, 4 was linearized by Restriction enzymes.  So, our restriction enzymes work correctly.
 
-
</div>
 
-
 
-
<div class="digestion lysis">
 
-
====Digestion of the PCR products of S gene by EcoRI and SpeI and <partinfo>E0840</partinfo> by EcoRI and XbaI to insert S gene to <partinfo>E0840</partinfo>====
 
-
{| class="experiments"
 
-
!Name||Sample Volume (&micro;l)||10×Buffer||Enzyme 1||Enzyme 2||MilliQ||Total||Incubation
 
-
|-
 
-
|S<sub>1</sub>||11||5||''EcoR''I 0.2||''Spe''I 0.2||33.6||50||rowspan="3"|At 37&#x2103; for 2h
 
-
|-
 
-
|S<sub>2</sub>||11||5||''EcoR''I 0.2||''Spe''I 0.2||33.6||50
 
-
|-
 
-
|<partinfo>E0840</partinfo>||45||5||''EcoR''I 0.2||''Xba''I 0.2||0||50
 
-
|}
 
-
After PCR purification, evaporated them and diluted 3ul.
 
-
</div>
 
-
 
-
<div class="ligation lysis">
 
-
====Ligation====
 
-
{| class="experiments"
 
-
!Name||Vector||Insert||Ligation High||Total
 
-
|-
 
-
|S-E0840<sub>1</sub>||<partinfo>E0840</partinfo> 0.5&micro;l||S<sub>1</sub> 0.5||1||2
 
-
|-
 
-
|S-E0840<sub>2</sub>||<partinfo>E0840</partinfo> 0.5||S<sub>2</sub> 0.5||1||2
 
-
|}
 
-
</div>
 
-
 
-
 
-
===Monday, July 26 <span class="by">By: Wataru, Tomonori, Makoto</span>===
 
-
<div class="electrophorersis lysis">
 
-
====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] of PCR Products====
 
-
[[Image:KyotoExp100726-1.png|300px|right]]
 
-
At the condition 4 (4.5&micro;l MgSO4) and 6 (6&micro;l MgSO4), S-R-Rz/Rz1 is amplified very much. So we decided to use them.
 
-
</div>
 
-
 
-
<div class="pcr-purification">
 
-
====PCR Purification====
 
-
{| class="experiments"
 
-
!No.||Concentration (ng/&micro;l)||New Name
 
-
|-
 
-
|4||51.6||SRRz<sub>1</sub>
 
-
|-
 
-
|5||59.3||
 
-
|-
 
-
|6||59.6||SRRz<sub>2</sub>
 
-
|}
 
-
</div>
 
-
 
-
<div class="transformation lysis">
 
-
====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
 
-
{| class="experiments"
 
-
!Name||Well||Sample (&micro;l)||Competent Cell (&micro;l)||Total (&micro;l)||Plate||Incubation||Result
 
-
|-
 
-
|<partinfo>E0240</partinfo>||1-12-M||1||20||21||LB (Amplicillin+)||rowspan="3"|At 37&#x2103; 7/26 - 7/27||×
 
-
|-
 
-
|<partinfo>I20260</partinfo>||2-17-F||1||20||21||rowspan="2"|LB (Kanamycin+)||×
 
-
|-
 
-
|<partinfo>J04450</partinfo>||1-5-E||1||20||21||×
 
-
|}
 
-
</div>
 
-
 
-
<div class="culture lysis measure">
 
-
====Culture of <partinfo>pSB4K5</partinfo>, <partinfo>E0840</partinfo>, and <partinfo>B0015</partinfo>====
 
-
</div>
 
-
===Tuesday, July 27 <span class="by">By: Wataru, Tomo, Kazuya, Ken, Naoi===
 
-
<div class="colony-pcr lysis">
 
-
====[[Team:Kyoto/Protocols#Colony_PCR~Colony PCR]] of S-E0840 (Electrophoresis for 35min)====
 
-
{|class="experiments"
 
-
|Marker||1||2||3||4||5||6||7||8||9||10||11||12||13||+||-||Marker
 
-
|-
 
-
|1kb||colspan="6"|S-E0840<sub>1</sub>||colspan="7"|S-E0840<sub>2</sub>||E0840||None||100bp
 
-
|}
 
-
[[Image:KyotoExp100727-1.png|300px|right]]
 
-
As a result, 1, 3, 5, 6, 11, 12, and 13 are inserted S gene correctly. So, we decided to use 6 as S-E0840<sub>1</sub> and 11 as S-E0840<sub>2</sub>.
 
-
</div>
 
-
 
-
<div class="miniprep lysis measure">
 
-
====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
 
-
{|class="experiments"
 
-
!Name||Concentration(ng/&micro;L)
 
-
|-
 
-
|<partinfo>R0011</partinfo>||26.9
 
-
|-
 
-
|<partinfo>B0015</partinfo>||120.0
 
-
|-
 
-
|<partinfo>E0840</partinfo>||120.1
 
-
|}
 
-
</div>
 
-
 
-
<div class="digestion lysis">
 
-
====[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]]====
 
-
{|class="experiments"
 
-
!||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total||Incubation
 
-
|-
 
-
|<partinfo>B0015</partinfo>||30||5||0.5||rowspan="3"|EcoRI||0.4||rowspan="3"|XbaI||0.3||13.7||50||rowspan="3"|At 37&#x2103; 16:45 - 18:00
 
-
|-
 
-
|SRRz<sub>1</sub>||40||5||0.5||0.4||0.4||3.8||50
 
-
|-
 
-
|SRRz<sub>2</sub>||40||5||0.5||0.4||0.4||3.8||50
 
-
|}
 
-
</div>
 
-
 
-
<div class="ligation lysis">
 
-
====[[Team:Kyoto/Protocols#Ligation|Ligation]]====
 
-
</div>
 
-
 
-
<div class="transformation lysis">
 
-
====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
 
-
{| class="experiments"
 
-
!Name||Sample (&micro;l)||Competent Cells (&micro;l)||Total (&micro;l)||Plate||Incubation||Result
 
-
|-
 
-
|SRRz<sub>1</sub>-B0015||||||||rowspan="2"|||rowspan="2"|||&#x25CB;
 
-
|-
 
-
|SRRz<sub>2</sub>-B0015||||||||&#x25CB;
 
-
|}
 
-
</div>
 
-
 
-
 
-
===Wednesday, July 28 <span class="by">By: </span>===
 
-
<div class="miniprep lysis">
 
-
====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
 
-
{| class="experiments"
 
-
!Name||Concentration(ng/&micro;l)
 
-
|-
 
-
|S-E0840<sub>1</sub>||95.5
 
-
|-
 
-
|S-E0840<sub>2</sub>||98.6
 
-
|}
 
-
Diluted S-E0840<sub>1</sub> and S-E0840<sub>2</sub> 20 times with water, and used as template DNA.
 
-
</div>
 
-
 
-
<div class="deletion-pcr lysis">
 
-
====[[Team:Kyoto/Protocols#Deletion_PCR|Deletion_PCR]] to delete a functional domain of S gene====
 
-
{| class="experiments"
 
-
!||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer Forward(10&micro;M)||Primer Reverse(10&micro;M)||Template S-E0840<sub>1</sub>||Template S-E0840<sub>2</sub>||KOD Plus ver.2||Total
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>1</sub>-1||28||3||5||5||1.5||1.5||5||-||1||50
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>1</sub>-2||28||3||5||5||1.5||1.5||5||-||1||50
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>2</sub>||28||3||5||5||1.5||1.5||-||5||1||50
 
-
|}
 
-
{|class="experiments"
 
-
|94&#x2103;||2min||
 
-
|-
 
-
|98&#x2103;||10sec||rowspan="3"|35 cycles
 
-
|-
 
-
|55&#x2103;||30sec
 
-
|-
 
-
|68&#x2103;||4min
 
-
|-
 
-
|4&#x2103;||forever||
 
-
|}
 
-
</div>
 
-
 
-
<div class="digestion lysis">
 
-
====Restriction Digestion to check the function of DpnI====
 
-
{| class="experiments"
 
-
!Name||Sample||fast digestion buffer||DpnI||MilliQ||Total
 
-
|-
 
-
|S-E0840<sub>1</sub>||3||1||0.1||5.8||10
 
-
|-
 
-
|S-E0840<sub>2</sub>||3||1||0.1||5.8||10
 
-
|}
 
-
</div>
 
-
 
-
<div class="electrophoresis">
 
-
====Electrophoresis for 35min====
 
-
{| class="experiments"
 
-
|Marker||1||2||3||4||Marker
 
-
|-
 
-
|1kb||Not digested S-E0840<sub>1</sub>||Not digested S-E0840<sub>2</sub>||Digested S-E0840<sub>1</sub>||Digested S-E0840<sub>2</sub>||100bp
 
-
|}
 
-
[[image:KyotoExp100728-1.png|300px|right]]
 
-
DpnI works correctly.
 
-
</div>
 
-
 
-
 
-
===Thursday, July 29 <span class="by">By: </span>===
 
-
<div class="digestion lysis">
 
-
====[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]]====
 
-
{| class="experiments"
 
-
!Name||Sample volume||Fastdigestion Buffer||colspan="2"|Enzyme 1||MilliQ||Total||Incubation
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>1</sub>-1||50||6||DpnI||0.2||3.8||60||rowspan="2"|07/29 09:40 - 07/29 11:00
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>2</sub>||50||6||DpnI||0.2||3.8||60
 
-
|}
 
-
</div>
 
-
 
-
<div class="ligation pospholylation lysis">
 
-
====[[Team:Kyoto/Protocols#Ligation|Ligation]] and [[Team:Kyoto/Protocols#Pospholylation|Pospholylation]]====
 
-
{|class="experiments"
 
-
!Name||Sample||MilliQ||Ligation High||T4 Kinase||Total||Incubation
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>1</sub>-1||2||7||5||1||15||rowspan="2"|07/29 11:30 ~ 07/29 13:00
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>2</sub>||2||7||5||1||15
 
-
|}
 
-
</div>
 
-
 
-
<div class="transformation lysis">
 
-
====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
 
-
{|class="experiments"
 
-
!Name||Sample Volume(&micro;L)||Competent Cell(&micro;L)||Total||Plate||Incubation||Result
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>1</sub>-1||3||30||33||rowspan="2"|LB Amp+||rowspan="2"|07/29 ~ 07/30||&#x25CB;
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>2</sub>||3||30||33||&#x25CB;
 
-
|}
 
-
</div>
 
-
 
-
 
-
===Monday, August 2 <span class="by">By: Wataru, Ken</span>===
 
-
<div class="miniprep lysis">
 
-
====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
 
-
{|class="experiments"
 
-
!Name||Concentration(ng/&micro;L)
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840-1||52.7
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840-2||54.4
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840-3||89.5
 
-
|-
 
-
|<partinfo>pSB4K5</partinfo>||50.7
 
-
|-
 
-
|<partinfo>R0011</partinfo>||18.6
 
-
|}
 
-
</div>
 
-
 
-
<div class="pcr measure">
 
-
====[[Team:Kyoto/Protocols#Standard_PCR|Standard PCR]] of <partinfo>E0240</partinfo>====
 
-
E240 is very important parts to measure RPU of promoters in iGEM. However, we failed to transfect it to E.coli from parts kit of iGEM. So we decided to amplify this parts by PCR.
 
-
{|class="experiments"
 
-
!Name||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10&micro;M)||Primer VR(10&micro;M)||Template E240||KOD Pllus ver.2||Total
 
-
|-
 
-
|E0240<sub>1</sub>||28||3||5||5||1.5||1.5||5||1||50
 
-
|-
 
-
|E0240<sub>2</sub>||28||3||5||5||1.5||1.5||5||1||50
 
-
|}
 
-
{|class="experiments"
 
-
|94&#x2103;||2min||
 
-
|-
 
-
|98&#x2103;||10sec||rowspan="3"|35 cycles
 
-
|-
 
-
|55&#x2103;||30sec
 
-
|-
 
-
|68&#x2103;||4min
 
-
|-
 
-
|4&#x2103;||forever||
 
-
|}
 
-
</div>
 
-
 
-
<div class="electrophoresis measure">
 
-
====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]]====
 
-
</div>
 
-
 
-
<div class="pcr-purification measure">
 
-
====PCR Purification====
 
-
{|class="experiments"
 
-
!Sample number||Concentration(ng/&micro;L)
 
-
|-
 
-
|E0240<sub>1</sub>||42.6
 
-
|-
 
-
|E0240<sub>2</sub>||55.3
 
-
|}
 
-
</div>
 
-
 
-
<div class="digestion measure">
 
-
====[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]] for inserting <partinfo>E0240</partinfo> to pSB4K5 by 3A assembly====
 
-
{| class="experiments"
 
-
!Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
 
-
|-
 
-
|E0240<sub>1</sub>(X-P)||30||5||0.5||XbaI||0.2||PstI||0.2||14.1||50
 
-
|-
 
-
|E0240<sub>2</sub>(X-P)||30||5||0.5||XbaI||0.2||PstI||0.2||14.1||50
 
-
|}
 
-
</div>
 
-
 
-
<div class="pcr-purication measure">
 
-
====PCR Purification====
 
-
{| class="experiments"
 
-
!Name||Concentration(ng/&micro;L)||Volume(&micro;L)
 
-
|-
 
-
|E0240<sub>1</sub>(X-P)||21.8||40
 
-
|-
 
-
|E0240<sub>2</sub>(X-P)||32.4||45
 
-
|}
 
-
Stored at -20&#x2103;.
 
-
</div>
 
-
 
-
<div class="error-pcr lysis">
 
-
====Error PCR====
 
-
{|class="experiments"
 
-
!Name||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10&micro;M)||Primer VR(10&micro;M)||Template &Delta;1||Template||Template||KOD Pllus ver.2||Total
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>1</sub>-1||32||3||5||5||1.5||1.5||1||-||-||1||50
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>1</sub>-2||32||3||5||5||1.5||1.5||-||1||-||1||50
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>2</sub>||32||3||5||5||1.5||1.5||-||-||1||1||50
 
-
|}
 
-
{|class="experiments"
 
-
|94&#x2103;||2min||
 
-
|-
 
-
|98&#x2103;||10sec||rowspan="2"|20 cycles
 
-
|-
 
-
|68&#x2103;||4min
 
-
|-
 
-
|4&#x2103;||forever||
 
-
|}
 
-
</div>
 
-
 
-
<div class="transformation lysis">
 
-
====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
 
-
{| class="experiments"
 
-
!Name||Sample (&micro;l)||Competent Cells (&micro;l)||Total (&micro;l)||Plate||Incubation||Result
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>1</sub>-1||2||20||22||rowspan="3"|||rowspan="3"|||&#x25CB;
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>1</sub>-2||2||20||22||&#x7D;
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>2</sub>||2||20||22||&#x25CB;
 
-
|}
 
-
</div>
 
-
 
-
 
-
===Tuesday, August 3 <span class="by">By: </span>===
 
-
<div class="culture lysis">
 
-
====Culture of each two colonies of S<sub>&Delta;TMD1</sub>-E0840<sub>1</sub>-1 and S<sub>&Delta;TMD1</sub>-E0840<sub>2</sub> for 37&#x2103; 08/03-08/04===
 
-
</div>
 
-
 
-
<div class="miniprep measure">
 
-
====[[Team:Kyoto/Protocols#Miniprep|Miniprep]] for Construction of Measure(''lac''P) and Measure(Standard)====
 
-
{|class="experiments"
 
-
!Sample number||Concentration(ng/&micro;L)
 
-
|-
 
-
|<partinfo>pSB4K5</partinfo>||60.7
 
-
|-
 
-
|<partinfo>R0011</partinfo>||26.8
 
-
|}
 
-
</div>
 
-
 
-
<div class="digestion measure">
 
-
====[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]]====
 
-
{|class="experiments"
 
-
!Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
 
-
|-
 
-
|R0011||50||6||0.6||EcoRI||0.2||SpeI||0.2||3||60
 
-
|-
 
-
|pSB4K5(E-P)||50||6||0.6||EcoRI||0.2||PstI||0.2||3||60
 
-
|-
 
-
|E0240<sub>1</sub>(X-P)||50||6||0.6||XbaI||0.2||PstI||0.2||3||60
 
-
|-
 
-
|E0240<sub>2</sub>(X-P)||50||6||0.6||XbaI||0.2||PstI||0.2||3||60
 
-
|}
 
-
</div>
 
-
 
-
<div class="pcr-purication">
 
-
====PCR Purification====
 
-
{|class="experiments"
 
-
!Sample number||Concentration(ng/&micro;L)
 
-
|-
 
-
|pSB4K5(E-P)||39.5
 
-
|-
 
-
|E0240<sub>1</sub>(X-P)||21.8
 
-
|-
 
-
|E0240<sub>2</sub>(X-P)||32.4
 
-
|}
 
-
pSB4K5(E-P) is concentrated 10&micro;L and E0240<sub>1</sub>(X-P), E0240<sub>2</sub>(X-P) are concentrated 1&micro;L.
 
-
</div>
 
-
 
-
<div class="ethanol-precipitation measure">
 
-
====Ethanol Precipitation====
 
-
</div>
 
-
 
-
<div class="measure">
 
-
====Dilution of <partinfo>pSB4K5</partinfo> by 2&micro;l MilliQ====
 
-
</div>
 
-
 
-
<div class="measure">
 
-
====[[Team:Kyoto/Protocols#Ligation|Ligation]]====
 
-
{|class="experiments"
 
-
!||Vector||rowspan="2"|Insert 1||rowspan="2"|Insert 2||Ligation High||Total||Incubation
 
-
|-
 
-
|R0011-E0240<sub>1</sub>[Low]||pSB4K5(E-P)||1||R0011(E-S)||1||E0240<sub>1</sub>(X-P)||1||3||15||colspan="2"|17:30 - 20:20
 
-
|-
 
-
|R0011-E0240<sub>2</sub>[Low]||pSB4K5(E-P)||1||R0011(E-S)||1||E0240<sub>2</sub>(X-P)||1||3||15
 
-
|}
 
-
</div>
 
-
 
-
<div class="measure">
 
-
====[[Team:Kyoto/Protocols#Standard PCR|Standard PCR]] of <partinfo>J23101</partinfo>-<partinfo>E0240</partinfo> that is important in the measurement of RPU====
 
-
{|class="experiments"
 
-
!Name||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10&micro;M)||Primer VR(10&micro;M)||Template J23101-E0240||KOD plus ver.2 ||Total
 
-
|-
 
-
|J23101-E0240<sub>1</sub>||32||3||5||5||1.5||1.5||1||1||50
 
-
|-
 
-
|J23101-E0240<sub>2</sub>||32||3||5||5||1.5||1.5||-||1||50
 
-
|}
 
-
{|class="experiments"
 
-
|94&#x2103;||2min||
 
-
|-
 
-
|98&#x2103;||10sec||rowspan="3"|30 cycles
 
-
|-
 
-
|55&#x2103;||30sec
 
-
|-
 
-
|68&#x2103;||4min
 
-
|-
 
-
|4&#x2103;||forever||
 
-
|}
 
-
</div>
 
-
 
-
<div class="measure-construction">
 
-
====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]====
 
-
{|class="experiments"
 
-
!Name||Concentration(ng/&micro;L)
 
-
|-
 
-
|J23101-E0240||40.6
 
-
|}
 
-
</div>
 
-
 
-
<div class="measure-construction">
 
-
====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]====
 
-
{|class="experiments"
 
-
!Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
 
-
|-
 
-
|J23101-E0240(E-P)||45||6||0.6||EcoRI||0.2||PstI||0.2||8||60
 
-
|}
 
-
</div>
 
-
 
-
<div class="measure-construction">
 
-
====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]====
 
-
{| class="experiments"
 
-
!Name||Concentration(ng/&micro;L)||Volume(&micro;L)
 
-
|-
 
-
|J23101-E0240(E-P)||74.1||30
 
-
|}
 
-
J23101-E0240(E-P) is concentrated 7&micro;L
 
-
</div>
 
-
 
-
<div class="measure-construction">
 
-
====[[Team:Kyoto/Protocols#Ligation|Ligation]]====
 
-
{|class="experiments"
 
-
!||Vector||colspan="2"|Insert||colspan="2"|Ligation High||Total||Incubation
 
-
|-
 
-
|J23101-E0240[Low]||pSB4K5(E-P)||1||J23101-E0240(E-P)||1||2||4||20:00-20:30
 
-
|}
 
-
</div>
 
-
 
-
<div class="measure-construction"><br />
 
-
====Transformation====
 
-
{| class="experiments"
 
-
!Name||Conc(/&micro;L)||Sample Volume(&micro;L)||Competent Cell(&micro;L)||Total||Plate||Incubation
 
-
|-
 
-
|R0011-E0240<sub>1</sub>[Low]||-||1||20||21||rowspan="3"|LB kan||rowspan="3"|8/3~8/4
 
-
|-
 
-
|R0011-E0240<sub>2</sub>[Low]||-||1||20||21
 
-
|-
 
-
|J23101-E0240[Low]||-||1||20||21
 
-
|}
 
-
</div>
 
-
 
-
 
-
===Thursday, August 5 <span class="by">By: </span>===
 
-
<div class="measure-construction">
 
-
====Result of Transformation====
 
-
{|class="experiments"
 
-
|R0011-E0240<sub>1</sub>[Low]||rowspan="3"|Many colonies
 
-
|-
 
-
|R0011-E0240<sub>2</sub>[Low]
 
-
|-
 
-
|J23101-E0240[Low]
 
-
|}
 
-
pSB4K5 is inserted RFP generator. We didn't distinguish this inserted parts from low copy plasmid backbone, so self-ligated colony is red. So, white colony is correctly inserted parts.
 
-
 
-
However, white colonies and green colonies are observed in R0011-E0240<sub>1</sub>[Low] and R0011-E0240<sub>2</sub>[Low] plate. We cultured both white and green colonies.
 
-
 
-
In J23101-E0240[Low], Many of colonies are red, but green colonies are observed. We cultured green colonies.
 
-
</div>
 
-
 
-
<div class="measure-constrcution">
 
-
{| class="experiments"
 
-
!Name||Color||Incubation
 
-
|-
 
-
|R0011-E0240<sub>1</sub>[Low]-1||Green Colony||rowspan="11"|8/5-8/6
 
-
|-
 
-
|R0011-E0240<sub>1</sub>[Low]-2||Green Colony
 
-
|-
 
-
|R0011-E0240<sub>1</sub>[Low]-3||White Colony
 
-
|-
 
-
|R0011-E0240<sub>1</sub>[Low]-4||White Colony
 
-
|-
 
-
|R0011-E0240<sub>2</sub>[Low]-1||Green Colony
 
-
|-
 
-
|R0011-E0240<sub>2</sub>[Low]-2||White Colony
 
-
|-
 
-
|R0011-E0240<sub>2</sub>[Low]-3||White Colony
 
-
|-
 
-
|R0011-E0240<sub>2</sub>[Low]-4||White Colony
 
-
|-
 
-
|J23101-E0240[Low]-1||Green Colony
 
-
|-
 
-
|J23101-E0240[Low]-2||Green Colony
 
-
|-
 
-
|J23101-E0240[Low]-3||Green Colony
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction sequence">
 
-
{| class="experiments"
 
-
!Name||Concentration(ng/&micro;L)
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>1</sub>-1-A||28.9
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>1</sub>-1-B||25.3
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>2</sub>-A||26.6
 
-
|-
 
-
|S<sub>&Delta;TMD1</sub>-E0840<sub>2</sub>-B||24.0
 
-
|}
 
-
As a result, deletion is succeeded, however, point mutation is failed. It is because DpnI is too little to digest all of template DNA.
 
-
</div>
 
-
 
-
 
-
===Friday, August 6===
 
-
<div class="measure-construction">
 
-
====Miniprep====
 
-
{| class="experiments"
 
-
!Name
 
-
|-
 
-
|R0011-E0240<sub>1</sub>[Low]-1
 
-
|-
 
-
|R0011-E0240<sub>1</sub>[Low]-2
 
-
|-
 
-
|R0011-E0240<sub>1</sub>[Low]-3
 
-
|-
 
-
|R0011-E0240<sub>1</sub>[Low]-4
 
-
|-
 
-
|R0011-E0240<sub>2</sub>[Low]-1
 
-
|-
 
-
|R0011-E0240<sub>2</sub>[Low]-2
 
-
|-
 
-
|R0011-E0240<sub>2</sub>[Low]-3
 
-
|-
 
-
|R0011-E0240<sub>2</sub>[Low]-4
 
-
|-
 
-
|J23101-E0240[Low]-1
 
-
|-
 
-
|J23101-E0240[Low]-2
 
-
|-
 
-
|J23101-E0240[Low]-3
 
-
|}
 
-
</div>
 
-
 
-
<div class="measure-construction">
 
-
====Restriction Digestion====
 
-
{|class="experiments"
 
-
!Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
 
-
|-
 
-
|R0011-E0240<sub>1</sub>[Low]-1||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
 
-
|-
 
-
|R0011-E0240<sub>1</sub>[Low]-2||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
 
-
|-
 
-
|R0011-E0240<sub>1</sub>[Low]-3||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
 
-
|-
 
-
|R0011-E0240<sub>1</sub>[Low]-4||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
 
-
|-
 
-
|R0011-E0240<sub>2</sub>[Low]-1||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
 
-
|-
 
-
|R0011-E0240<sub>2</sub>[Low]-2||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
 
-
|-
 
-
|R0011-E0240<sub>2</sub>[Low]-3||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
 
-
|-
 
-
|R0011-E0240<sub>2</sub>[Low]-4||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
 
-
|-
 
-
|J23101-E0240[Low]-1||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
 
-
|-
 
-
|J23101-E0240[Low]-2||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
 
-
|-
 
-
|J23101-E0240[Low]-3||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
 
-
|}
 
-
</div>
 
-
 
-
<div class="measure-construction">
 
-
=====Electrophoresis=====
 
-
{| class="experiments"
 
-
|M||M||M||M||1||2||3||4||5||6||7||8||9||10||11||12||13
 
-
|-
 
-
|100bp||&lambda;||&lambda;||100bp
 
-
|J23101-E0240[Low]-1||J23101-E0240[Low]-2||J23101-E0240[Low]-1
 
-
|R0011-E0240<sub>1</sub>[Low]-1||R0011-E0240<sub>1</sub>[Low]-2||R0011-E0240<sub>1</sub>[Low]-3||R0011-E0240<sub>1</sub>[Low]-4
 
-
|R0011-E0240<sub>2</sub>[Low]-1||R0011-E0240<sub>2</sub>[Low]-2||R0011-E0240<sub>2</sub>[Low]-3||R0011-E0240<sub>2</sub>[Low]-4
 
-
|J23101-E0240[Low]-1||J23101-E0240[Low]-2
 
-
|}
 
-
[[Image:KyotoExp100806-1.png]]
 
-
J23101-E0240[Low]-1, J23101-E0240[Low]-1, R0011-E0240<sub>1</sub>[Low]-1, R0011-E0240<sub>1</sub>[Low]-2, R0011-E0240<sub>2</sub>[Low]-1 are inserted correctly. White colonies are not inserted <partinfo>R0011</partinfo> but its vector. Top10 we used are deleted Lac operon. Then, correctly inserted parts is green because of the lack of ''lacI''.
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Error PCR (Retry)====
 
-
{| class="experiments"
 
-
!Name||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10&micro;M)||Primer VR(10&micro;M)||Template &Delta;TMD failed(50ng/&micro;L)||KOD plus ver.2||Total
 
-
|-
 
-
|&Delta;TMD①||32||3||5||5||1.5||1.5||1||1||50
 
-
|-
 
-
|&Delta;TMD②||32||3||5||5||1.5||1.5||1||1||50
 
-
|}
 
-
{|class="experiments"
 
-
|94&#x2103;||2min
 
-
|-
 
-
|98&#x2103;||10sec||rowspan="2"|25 cycles
 
-
|-
 
-
|68&#x2103;||4min
 
-
|-
 
-
|colspan="2"|Add DpnI 2&micro;l
 
-
|-
 
-
|Incubate||1h
 
-
|-
 
-
|4&#x2103;||forever||
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Transformation====
 
-
{| class="experiments"
 
-
!Name||Conc(/&micro;L)||Sample Volum(&micro;L)||Competent Cell(&micro;L)||Total||Plate||Incubation
 
-
|-
 
-
|&Delta;TMD①||-||4||50||54||rowspan="3"|LB kan||rowspan="4"|8/6~8/9
 
-
|-
 
-
|&Delta;TMD②||-||4||50||54
 
-
|-
 
-
|2-17-F||-||2||50||52
 
-
|-
 
-
|2-I-5||||2||50||52||LB amp
 
-
|}
 
-
</div>
 
-
 
-
 
-
===Monday, August 9 <span class="by">By: Wataru, Tomonori, Ken, Takuya</span>===
 
-
<div class="measure-construction">
 
-
====Miniprep of MS and ML====
 
-
{|class="experiments"
 
-
!Sample number||concentration(ng/&micro;L)
 
-
|-
 
-
|MS||116.2
 
-
|-
 
-
|ML||146.6
 
-
|}
 
-
</div>
 
-
 
-
<div class="measure-construction">
 
-
====Transfotrmation of MS and ML====
 
-
{|class="experiments"
 
-
!Sample||conc(ng/&micro;L)||Sample vol(&micro;L)||Competent Cell||Competent cell vol(&micro;L)||Total vol(&micro;L)||Plate||Incuvation
 
-
|-
 
-
|MS||116.2||2||KRX||50||52||rowspan="2"|LB kanamycin||rowspan="2"|8/9 18:00‾8/10 12:00
 
-
|-
 
-
|ML||146.6||2||KRX||50||52
 
-
|}
 
-
</div>
 
-
 
-
<div class="measure-construction">
 
-
====Restriction enzyme digestion and ethanol precipitation====
 
-
To use lac p for next ligation, we digested 1-6-G by EroRI and PstI
 
-
{|class="experiments"
 
-
!Sample||10x Buffer||BSA||Enzyme (EcoRI)||Enzyme (PstI)||MilliQ||Total
 
-
|-
 
-
|50||6||0.6||0.5||0.5||2.4||60
 
-
|}
 
-
 
-
Incubate 37&#x2103; 8/9 16:20‾18:20
 
-
 
-
After restriction enzyme digestion, we did ethanol precipitation.
 
-
</div>
 
-
 
-
<div class="measure-construction">
 
-
====Ligation and Transformation====
 
-
{|class="experiments"
 
-
!Sample||Conc (nu/&micro;L)||Sample vol (&micro;L)||Competent cell||Competent cell vol (&micro;L)||Total vol (&micro;L)||Plate||Incuvation
 
-
|-
 
-
|rowspan="2"|Lac p (low)||rowspan="2"|-||2||KRX||50||52||rowspan="2"|LB kanamycin||rowspan="2"|8/9 20:00‾8/10 9:00
 
-
|-
 
-
|2||C2||50||52
 
-
|}
 
-
</div>
 
-
 
-
 
-
===Tuesday, August 10 <span class="by">By: Wataru, Tomonori, Ken, Fumitaka</span>
 
-
<div class="measure-construction">
 
-
====Making culture plate on lac p (low), MS and ML====
 
-
{|class="experiments"
 
-
|rowspan="2"|Lac p (low)||KRX||rowspan="4"|Many colonies
 
-
|-
 
-
|C2
 
-
|-
 
-
|MS||KRX
 
-
|-
 
-
|ML||KRX
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Minprep of &Delta;TMD1+GFP====
 
-
{|class="experiments"
 
-
!Sample number||Concentration (ng/&micro;L)
 
-
|-
 
-
|1-1||9.9
 
-
|-
 
-
|1-2||27.3
 
-
|-
 
-
|2-1||43.2
 
-
|-
 
-
|2-2||34.7
 
-
|}
 
-
37&#x2103; 8/9 18:00‾8/10 9:00
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Culture and Master Plate====
 
-
</div>
 
-
 
-
 
-
===Wednesday, August 11 <span class="by">By: Wataru, Naoi, Ken, Takuya</span>
 
-
<div class="lysis-construction">
 
-
{| class="experiments"
 
-
!Sample||Medium||Cloud||Incubation
 
-
|-
 
-
|rowspan="2"|1||Kanamycin||o||rowspan="14"|37&#x2103;8/10 20:00‾8/11 9:00
 
-
|-
 
-
|Ampicillin||x
 
-
|-
 
-
|rowspan="2"|2||Kanamycin||o
 
-
|-
 
-
|Ampicillin||o
 
-
|-
 
-
|rowspan="2"|3||Kanamycin||o
 
-
|-
 
-
|Ampicillin||x
 
-
|-
 
-
|rowspan="2"|4||Kanamycin||o
 
-
|-
 
-
|Ampicillin||x
 
-
|-
 
-
|rowspan="2"|5||Kanamycin||o
 
-
|-
 
-
|Ampicillin||x
 
-
|-
 
-
|rowspan="2"|6||Kanamycin||o
 
-
|-
 
-
|Ampicillin||o
 
-
|-
 
-
|rowspan="2"|7||Kanamycin||o
 
-
|-
 
-
|Ampicillin||x
 
-
|}
 
-
 
-
Discussion: About sample 1, 3, 4, 5 and 7, lac promoter was correctly inserted in low copy plasmid. About sample 2 and 6, low copy plasmid and vector derived from lac promoter were ligated. We decided to use sample 1 or 3.
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Miniprep of C2+lac(low), S-R-Rz 1', 3'====
 
-
lac(low)1 : 31.2 (ng/&micro;L)
 
-
lac(low)2 : 29.9 (ng/&micro;L)
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Restriction Digestion and electrophoresis of lac (low) 1 and 3====
 
-
{| class="experiments"
 
-
!Name||EcoRI||PstI
 
-
|-
 
-
|1||0.2||-
 
-
|-
 
-
|2||-||0.2
 
-
|-
 
-
|3||0.2||0.2
 
-
|-
 
-
|N||-||-
 
-
|}
 
-
Sample: 1-1, 1-2, 1-3, 1-N, 3-1, 3-2, 3-3, 3-N
 
-
 
-
M 1-1 1-2 1-3 1-N M  M 3-1 3-2 3-3 3-N M
 
-
 
-
[[image:KyotoExp100811-1.png]]
 
-
 
-
Discussion: Each enzyme correctly cut samples.
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Screening PCR of SRRz====
 
-
Sample: 1-20
 
-
 
-
Control: P(1-23L) P'(2-8E) N
 
-
 
-
Maker: lambda
 
-
 
-
M  N  P  P' P  1  2  3  4  5  6  M
 
-
 
-
[[image:KyotoExp100811-2.png]]
 
-
 
-
7 8 9 10 11 12 13 M 14 15 16 18 19 20 M
 
-
 
-
[[image:KyotoExp100811-3.png]]
 
-
 
-
Discussion: All of the sample were self-ligation of DT and SRRz weren't inserted.
 
-
</div>
 
-
 
-
 
-
===Thursday, August 12 <span class="by">By: Wataru, Ken</span>===
 
-
<div class="lysis-construction">
 
-
====Restriction Digestion and electrophoresis of <partinfo>B0015</partinfo>====
 
-
{| class="experiments"
 
-
!Sample name||Template||10xbuffer||100xbuffer||EcoRI||XbaI 1||XbaI 2||SpeI||PstI 1||PstI 2||Water||Total
 
-
|-
 
-
|1||3||1||0.1||0.2||-||-||-||-||-||5.7||10
 
-
|-
 
-
|2||3||1||0.1||-||0.2||-||-||-||-||5.7||10
 
-
|-
 
-
|3||3||1||0.1||-||-||0.2||-||-||-||5.7||10
 
-
|-
 
-
|4||3||1||0.1||-||-||-||0.2||-||-||5.7||10
 
-
|-
 
-
|5||3||1||0.1||-||-||-||-||0.2||-||5.7||10
 
-
|-
 
-
|6||3||1||0.1||-||-||-||-||-||0.2||5.7||10
 
-
|-
 
-
|N||3||1||0.1||-||-||-||-||-||-||5.9||10
 
-
|}
 
-
 
-
Sample: 1-6, N Maker: lambda, 100
 
-
 
-
M 1 2 3 4 5 6 N M M M
 
-
 
-
[[image:KyotoExp100812-1.png]]
 
-
 
-
Discussion: Each enzyme correctly cut each sample and was active.
 
-
</div>
 
-
 
-
 
-
===Thursday, August 19 <span class="by">By: Wataru, Tomo, Ken</span>
 
-
<div class="lysis-construction">
 
-
====Miniprep of S&Delta;TMD1GFP====
 
-
29.6(ng/&micro;g)
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Point mutation PCR of &Delta;TMD1GFP====
 
-
{| class="experiments"
 
-
!Sample number||Template||10xbuffer||dNTPs||MgSO4||Primer 1||Primer 2||Water||KOD-plus-||Total
 
-
|-
 
-
|1||1.5||5||5||3||1.5||1.5||31.5||1||50
 
-
|-
 
-
||2||1.5||5||5||3||1.5||1.5||31.5||1||50
 
-
|-
 
-
|control||1.5||5||5||3||1.5||1.5||32.5||-||50
 
-
|}
 
-
{| class="experiments"
 
-
|94(&#x2103;)||2min||
 
-
|-
 
-
|98||10sec||rowspan="3"|30cycles
 
-
|-
 
-
|55||30sec
 
-
|-
 
-
|68||3.5min
 
-
|-
 
-
|4.0||forever||
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Restriction Digestion(DpnI): 17:50-18:50====
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Electrophoresis====
 
-
Sample: 1, 2, Control Marker: lambda, 100
 
-
[[Image:KyotoExp100819-1.png]]
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Ligation and Transformation====
 
-
We named point mutation PCR products r&Delta;TMD1GFP.
 
-
</div>
 
-
 
-
 
-
===Monday, August 23 <span class="by">By: Wataru, Tomo, Ken, Fumitaka, Tasuku</span>===
 
-
<div class="lysis-construction">
 
-
====Miniprep of &Delta;TMD1====
 
-
{| class="experiments"
 
-
|Sample number||Concentration(ng/&micro;g)
 
-
|-
 
-
|1-1||58.9
 
-
|-
 
-
|2-2||49.9
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Sequencing of &Delta;TMD1 and MS====
 
-
Sample: r&delta;TMD1GFP1-1, 2-2, and MS
 
-
 
-
Discussion: The sequencing was in success and the results were desirable. It meant point mutation of &delta;TMD1GFP was succeeded and sequence of MS was confirmed. We decided to use r&delta;TMD1GFP.
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Screening PCR of SRRz-DT====
 
-
Sample: 1-13, Marker: lambda and 100, Control:P(1-23L) and N
 
-
 
-
{| class="experiments"
 
-
|90&#x2103;||10min||
 
-
|-
 
-
|94&#x2103;||30sec||rowspan="3"|35cycles
 
-
|-
 
-
|50&#x2103;||30sec
 
-
|-
 
-
|72&#x2103;||1.5min
 
-
|-
 
-
|72&#x2103;||4min||
 
-
|-
 
-
|4&#x2103;||hold||
 
-
|}
 
-
 
-
M  1  2  3  4  5  6  7  8  9  10 11 12 13  P  N  M
 
-
 
-
[[Image:KyotoExp100823-1.png]]
 
-
 
-
Discussion: We found the band; about 200bp, and it meant the lligation was completed successfully.
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Deletion PCR of r&Delta;TMD1GFP 2-2====
 
-
{| class="experiments"
 
-
!Sample||10x||dNTPs||Primer1||Primer2||Template||Water||KOD-plus-||Total
 
-
|-
 
-
|1||5||5||1.5||1.5||1||35||1||50
 
-
|-
 
-
|2||5||5||1.5||1.5||1||35||1||50
 
-
|-
 
-
|Control||5||5||1.5||1.5||1||35||-||50
 
-
|}
 
-
{| class="experiments"
 
-
|94&#x2103;||2min||
 
-
|-
 
-
|94&#x2103;||10sec||rowspan="3"|35cycles
 
-
|-
 
-
|56&#x2103;||30sec
 
-
|-
 
-
|68&#x2103;||3.5min
 
-
|-
 
-
|4&#x2103;||hold||
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Restriction Digestion(DpnI)====
 
-
{| class="experiments"
 
-
|Template||25(&micro;L)
 
-
|-
 
-
|DpnI||1
 
-
|-
 
-
|Total||26
 
-
|}
 
-
19:10-20:10
 
-
</div>
 
-
 
-
<div class="lysis-contruction">
 
-
====Ligation====
 
-
{|class="experiments"
 
-
!Sample Template||Water||Ligation high||T4 Kinase||total
 
-
|-
 
-
|1||3||6||5||1||15
 
-
|-
 
-
|2||3||6||5||1||15
 
-
|-
 
-
|Control||3||6||5||1||15
 
-
|}
 
-
20:15-21:15
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Transformation====
 
-
We named sample 1, 2 and control rr&delta;TMD1GFP1, 2 and control.
 
-
</div>
 
-
 
-
 
-
===Tuesday, August 24 <span class="by">By:Ken, Tomo, Tasuku, Takuya</span>===
 
-
<div class="lysis-construction">
 
-
====Retry of deletion PCR of r&delta;TMD1 GFP====
 
-
{| class="experiments"
 
-
!Sample||10x||dNTPs||MgSO4||Primer1||Primer2||Template||Water||KOD-plus-||Total
 
-
|-
 
-
|1||5||5||3||1.5||1.5||1||32||1||50
 
-
|-
 
-
|2||5||5||3||1.5||1.5||1||32||1||50
 
-
Control||5||5||3||1.5||1.5||1||32||1||50
 
-
|}
 
-
{|class="experiments"
 
-
|94&#x2103;||2min||
 
-
|-
 
-
|94&#x2103;||10sec||rowspan="3"|35cycles
 
-
|-
 
-
|58&#x2103;||30sec
 
-
|-
 
-
|68&#x2103;||3.5min
 
-
|-
 
-
|4&#x2103;||hold||
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Restriction Digestion (DpnI)====
 
-
14:15-15:15
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Electrophoreis====
 
-
Sample: 1, 2, and control, Maker: 100 and lambda
 
-
M    1  2  C        M
 
-
 
-
[[Image:KyotoExp100824-1.png]]
 
-
 
-
We found the band of sample 1 and 2 about 3000bp and there wasn't the band of sample control. So, we confirmed the PCR and RE were completed successfully.
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Ligation====
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Point mutation of SRRz====
 
-
{| class="experiments"
 
-
!Sample||10x||dNTPs||MgSO4||Primer1||Primer2||Template||Water||KOD-plus-||total
 
-
|-
 
-
|1||5||5||3||1.5||1.5||1||32||1||50
 
-
|-
 
-
|2||5||5||3||1.5||1.5||1||32||1||50
 
-
|-
 
-
|control||5||5||3||1.5||1.5||1||32||1||50
 
-
|-
 
-
|}
 
-
{|class="experiments"
 
-
|94&#x2103;||2min||
 
-
|-
 
-
|98&#x2103;||10sec||rowspan="3"|30cycles
 
-
|-
 
-
|55&#x2103;||30sec
 
-
|-
 
-
|68&#x2103;||4min
 
-
|-
 
-
|4&#x2103;||hold||
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Restriction Digestion(DpnI), electrophoresis and ligation====
 
-
[[Image:KyotoExp100824-2.png]]
 
-
 
-
We could find point mutation PCR and restriction enzyme of DpnI was done.
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====PCR of E0240===
 
-
{| class="experiments"
 
-
!Sample||10×||dNTPs||MgSO4||VF2||VR||Template||Water||KOD-plus-||Total
 
-
|-
 
-
|1||5||5||3||1.5||1.5||1||31.5||1||50
 
-
|-
 
-
|2||5||5||3||1.5||1.5||1||31.5||1||50
 
-
|-
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====PCR Purification===
 
-
Sample1: 5.5*50(ng/&micro;L)
 
-
Sample2: 5.2*50(ng/&micro;L)
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Restriction Digestion(EcoRI, PstI) and Gel extraction====
 
-
Sample1: 28.8 (ng/&micro;L)
 
-
Sample2: 26.4 (ng/&micro;L)
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Transformation====
 
-
Sample: rr&Delta;TMD1GFP1. 2. control, and rSRRz1. 2. control
 
-
</div>
 
-
 
-
 
-
===Wednesday, August 25 <span class="by">By:Ken, Tomo, Kazuya, Tasuku, Takuya<span>===
 
-
<div class="lysis-construction">
 
-
====Making culture and Master plate====
 
-
{| class="experiments"
 
-
|rr&Delta;TMD1-1||rowspan="2"|Many Colonies
 
-
|-
 
-
|rr&Delta;TMD1-2
 
-
|-
 
-
|rr&Delta;TMD1-C-||zero
 
-
|-
 
-
|rSRRz-1||rowspan="2"|Many Colonies
 
-
|-
 
-
|rSRRz-2
 
-
|-
 
-
|rSRRz-C-||zero
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Miniprep of 1-5G====
 
-
29.0 (ng/&micro;L)
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Restriction Digestion and purification of 1-5G(low copy plasmid) and lac low====
 
-
{| class="experiments"
 
-
!Sample name||Template||10xbuffer||100xbuffer||EcoRI||SpeI||PstI||Water||Total
 
-
|-
 
-
|1-5G||50||6||0.6||0.4||0.4||-||2.6||60
 
-
|-
 
-
|Lac low||10||4||0.4||-||0.3||0.3||25||40
 
-
|}
 
-
{|class="experiments"
 
-
|Sample Name||Concentration(ng/&micro;L)
 
-
|-
 
-
|1-5G||18.4
 
-
|-
 
-
|Lac low||8.6
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Ligation of <partinfo>E0240</partinfo> and <partinfo>pSB4K5</partinfo>, Transformation====
 
-
</div>
 
-
 
-
 
-
===Thursday, August 26 <span class="by">By:Ken, Tomo, Kazuya, Tasuku, Takuya, Fumitaka</span>
 
-
<div class="lysis-construction">
 
-
====Miniprep====
 
-
{| class="experiments"
 
-
|Sample name||Concentration(ng/&micro;L)
 
-
|-
 
-
|constP(0.7)||44.5
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Restriction Digestion of constP(0.7)====
 
-
{| class="experiments"
 
-
!Template||10xbuffer||100xbuffer||SpeI||PstI||Water||Total
 
-
|-
 
-
|25||4||0.4||0.3||0.3||10||40
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Purification of constP (0.7)====
 
-
49.8 ng/&micro;L
 
-
</div>
 
-
 
-
 
-
===Friday, August 27 <span class="by">By:Ken, Tomo, Kazuya, Fumitaka</span>===
 
-
<div class="lysis-construction">
 
-
 
-
====Making master plate of E0240 low====
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
{| class="experiments"
 
-
|Sample Name||Concentration(ng/&micro;L)
 
-
|-
 
-
|rr&Delta;TMD1 1-2||20.9
 
-
|-
 
-
|rSRRz 1-1||16.4
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Restriction Digestion of rr&Delta;TMD1 and rSRRz====
 
-
{| class="experiments"
 
-
!Sample name||Template||10xbuffer||100xbuffer||XbaI||PstI||Water||Total
 
-
|-
 
-
|rr&Delta;TMD1 1-2||45||6||0.6||0.3||0.3||7.8||60
 
-
|-
 
-
|rSRRz 1-1||45||6||0.6||0.3||0.3||7.8||60
 
-
|}
 
-
(13:20-14:20)
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Purification====
 
-
{|Sample Name||Concentration(ng/&micro;L)
 
-
|-
 
-
|rr&Delta;TMD1 1-2||44.7
 
-
|-
 
-
|rSRRz 1-1||56.1
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Lagation and transformation====
 
-
lacP + rr&Delta;TMD1 1-2
 
-
constP (0.7) + rr&Delta;TMD1 1-2
 
-
lac low + rSRRz 1-1
 
-
</div>
 
-
 
-
 
-
===Monday, August 30 <span class="by">By: Tomonori, Kazuya, Tasuku, Ken</span>===
 
-
<div class="lysis-construction">
 
-
====Making culture and Master plate====
 
-
{|class="experiments"
 
-
|lacP rr&Delta;TMD1GFP||Many colonies
 
-
|-
 
-
|lacP rr&Delta;TMD1GFP(control)||Some colonies
 
-
|-
 
-
|constP rr&Delta;TMD1GFP||Many colonies
 
-
|-
 
-
|constP rr&Delta;TMD1GFP(control)||Many colonies
 
-
|-
 
-
|lacP rSRRz low||No colony
 
-
|-
 
-
|lacP rSRRz low(control)||No colony
 
-
|}
 
-
 
-
Discussion: There ware some colonies, which emitted green light, on the plate 1.  So, we cultured those colonies on master plate.
 
-
On the plate 5 and 6, even though we used KRX, which is able to repress lac promoter, colonies might be dead.  However, we still have to do some experience so that we confirm lac promoter cannot repress enough and E. coli cannot survive.
 
-
</div>
 
-
 
-
 
-
===Tuesday, August 31 <span class="by">By: Tomonori, Takuya Y., Kazuya, Tasuku, Takuya, Ken<span>
 
-
<div class="lysis-construction">
 
-
====Miniprep====
 
-
{|class="experiments"
 
-
|constP (0.3)||48.5 (ng/&micro;L)
 
-
|-
 
-
|lac rr&Delta;TMD1||107.3
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====RE of constP (0.3) and lac rr&Delta;TMD1====
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Gel Extraction of lac rr&Delta;TMD1====
 
-
[[image:KyotoExp100831-1.png]]
 
-
 
-
45min
 
-
 
-
Discussion: There were two band at the bottom of the gel.  It was too long -45min-, and insert and vector might be contaminated.  But we went on next operation.
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Purification of constP (0.3) and lac rr&Delta;TMD1====
 
-
{|class="experiments"
 
-
|constP (0.3)||5.8 (ng/&micro;L)
 
-
|-
 
-
|lac rr&Delta;TMD1||7.8 (ng/&micro;L)
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Ligation and transformation====
 
-
{|class="experiments"
 
-
|Insert||Vector
 
-
|-
 
-
|lac rr&Delta;TMD1||constP (0.3)
 
-
|}
 
-
</div>
 
-
 
-
 
-
===Wednesday, September 1 <span class="by">By: Tomonori, Kazuya, Tasuku, Fumitaka, Ken</span>
 
-
<div class="lysis-construction">
 
-
====Making culture and Master plate====
 
-
{| class="experiments"
 
-
|lac rr&Delta;TMD1 constP||many colonies
 
-
|-
 
-
|lac rr&Delta;TMD1 const (control)||many colonies
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
Screenig PCR of lacP-rr&Delta;TMD1GFP-constP
 
-
Sample: 1-13 Control: Positive (1-23L) Maker: lambda, 100
 
-
 
-
    M  1  2  3  4  5  6  7    8  9  10 11 12  13  P M
 
-
 
-
[[image:KyotoExp100901.png]]
 
-
 
-
Discussion: All of the sample except sample 10 might be self-ligation products of constP.
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Miniprep====
 
-
{|class="experiments"
 
-
|rSRRz 1-1||33.8 (ng/&micro;L)
 
-
|-
 
-
|low||56.0 (ng/&micro;L)
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Restriction Digestion of rSRRz and low====
 
-
{|class="experiments"
 
-
!Sample name||Template||10xbuffer||100xbuffer||EcoRI||PstI||Water||Total
 
-
|-
 
-
|rSRRz||20||4||0.4||0.3||0.3||15||40
 
-
|-
 
-
|low||20||4||0.4||0.3||0.3||15||40
 
-
|}
 
-
(13:25-14:30)
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Purification====
 
-
{|class="experiments"
 
-
|rSRRz||6.5 (ng/&micro;L)
 
-
|-
 
-
|low||16.8
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Ligation and transformation====
 
-
Insert: rSRRz 1-1 Vector: low copy plasmid
 
-
</div>
 
-
 
-
 
-
===Thursday, September 2 <span class="by">By: Tomonori, Tomo, Takuya, Ken<span>===
 
-
<div class="lysis-construction">
 
-
====Making culture and Master plate====
 
-
{|class="experiments"
 
-
|rSRRz low||13 colonies
 
-
|-
 
-
|rSRRz low (Control)||13colonies
 
-
|}
 
-
</div>
 
-
 
-
<div class="lysis-construction">
 
-
====Screening PCR of rSRRz low====
 
-
Sample: rSRRz (1‾13)
 
-
Maker: lambda, 100
 
-
Control: Positive (1-23L), Neganive
 
-
 
-
    M  1  2  3  4  5  6  7  8  9  10  11 12 13  P  N  M
 
-
 
-
[[image:KyotoExp100902.png]]
 
-
 
-
Discussion: From sample 1, two vectors might be ligated.  Sample 3 and 4, rSRRz might be inserted in low copy plasmid correctly.  Sample 11, it might be the self-ligation product of low copy plasmid.  Anyway, we decided to culture those 4 colonies on master plate.
 
-
</div>
 
-
 
-
 
-
===Friday, September 3 <span class="by">By: Tomonori, Tomo, Kazuya, Tasuku, Fumitaka, Ken</span>===
 
-
<div class="lysis-construction">
 
-
====Making culture====
 
-
lac rr&Delta;TMD1 1, 3
 
-
rr&Delta;TMD1 1-1, 1-2
 
-
rSRRz 1-1, 1-2
 
-
ML
 
-
</div>
 
----
----

Latest revision as of 11:33, 27 October 2010

Contents

Notebook

Notebooks

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Other Information

  • Protocols: Protocols of each experiment such as Polymerase Chain Reaction (PCR), Restriction Digestion, Ligation, Transformation.
  • Materials: Strains, DNA, and Primers.
  • Parts: Construction of each part and BioBrick Parts used in our project.

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Retrieved from "http://2010.igem.org/Team:Kyoto/Notebook"