Team:Kyoto/Notebook

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{{:Team:Kyoto/Header}}
{{:Team:Kyoto/Header}}
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==Index==
 
==Notebook==
==Notebook==
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<div id="note">
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===Notebooks===
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===Tuesday, July 20 <span class="by">By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto</span>===
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* [[Team:Kyoto/Notebook1|Notebook1]]: Construction for Lysisbox.
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<div class="preparation">
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* [[Team:Kyoto/Notebook2|Notebook2]]: Measurement of R0011.
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====Solubilization of Antibiotics====
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* [[Team:Kyoto/Notebook3|Notebook3]]: Measurement of Lysis Cassette and Lysisbox etc.
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{| class="experiments"
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|Ampicillin(Amp)||Kanamycin(Kan)||
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-
|-
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|Mix 1.0g Amp and 20ml MilliQ||Mix 0.5g Kan and 10ml MilliQ||Final concentration is 50mg/ml
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|-
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|colspan="2"|Dispense 1.1ml of the solution into 1.5ml tubes||
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|-
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|colspan="2"|Store in the freezer (-20&#x2103;)||
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|}
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</div>
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<!---->
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<div class="plate">
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====Making plates for LB (Amp+) and LB (Kan+)====
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-
</div>
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<!---->
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<div class="transformation lysis measure">
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====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
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{| class="experiments"
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!Name||Well||Sample (&micro;l)||Competent Cells (&micro;l)||Total (&micro;l)||Plate||Incubation||Result
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|-
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|<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;
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-
|-
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|<partinfo>J23105</partinfo>||1-18-M||1||20||21||&#x25CB;
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|-
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|<partinfo>J23116</partinfo>||1-20-M||1||20||21||&#x25CB;
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|-
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|<partinfo>R0011</partinfo>||1-6-G||1||20||21||&#x25CB;
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|-
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|<partinfo>E0840</partinfo>||1-12-O||1||20||21||&#x25CB;
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|-
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|<partinfo>J06702</partinfo>||2-8-E||1||20||21||&#x25CB;
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|-
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|<partinfo>pSB4K5</partinfo>||1-5-G||1||20||21||&#xD7;
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|-
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|<partinfo>B0015</partinfo>||1-23-L||1||20||21||LB (Kan+)||&#xD7;
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|}
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A vector of "pSB4K5" 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 "pSB4K5" and "B0015".
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</div>
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 +
[[#top-section|^Top]]
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===Wednesday, July 21 <span class="by">By: Wataru, Ken, Makoto, Takuya Y.</span>===
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===Other Information===
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<div class="culture lysis measure">
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* [[Team:Kyoto/Protocols|Protocols]]: Protocols of each experiment such as Polymerase Chain Reaction (PCR), Restriction Digestion, Ligation, Transformation.
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====Culture of plates in which colonies was observed at 37&#x2103; from 07/21 20:50 to 07/22 17:00====
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* [[Team:Kyoto/Materials|Materials]]: Strains, DNA, and Primers.
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</div>
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* [[Team:Kyoto/Parts|Parts]]: Construction of each part and BioBrick Parts used in our project.
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<!---->
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<div class="master lysis measure">
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====Making a master plate of the above plates====
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</div>
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<!---->
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<div class="transformation lysis measure">
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====Retry [[Team:Kyoto/Protocols#Transformation|Transformation]]====
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{| class="experiments"
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!Name||Well||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
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|-
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|<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||○
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|-
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|<partinfo>B0015</partinfo>||1-23-L||1||20||21||○
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|}
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</div>
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<!---->
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<div class="pcr lysis">
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====[[Team:Kyoto/Protocols#Stantard_PCR|PCR]] for S-R-Rz/Rz1 and S====
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{| class="experiments"
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!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
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|-
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|1||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
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|-
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|2||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
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|-
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|3||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
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|-
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|4||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
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|-
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|5||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
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|-
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|6||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
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|-
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|7||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
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|-
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|8||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
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|}
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{|class="experiments"
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|-
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|94&#x2103;||2min||
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|-
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|98&#x2103;||10sec||rowspan="3"|30 cycles
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|-
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|55&#x2103;||30sec
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-
|-
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|68&#x2103;||4min
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-
|-
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|4&#x2103;||forever||
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|}
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</div>
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 +
[[#top-section|^Top]]
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===Thursday, July 22 <span class="by">By: Wataru</span>===
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----
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<div class="electrophoresis lysis">
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====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] of the PCR products for 40min====
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[[Image:KyotoExp100722-1.png|300px|right]]
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Length of S and S-R-Rz/Rz1 is 370bp and 1300bp, so PCR succeeded.
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</div>
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<!---->
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<div class="miniprep lysis measure">
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====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
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{| class="experiments"
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!Name||Concentration(ng/&micro;l)
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|-
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|<partinfo>J23100</partinfo>||18.5
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|-
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|<partinfo>J23105</partinfo>||12.5
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|-
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|<partinfo>J23116</partinfo>||14.6
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|-
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|<partinfo>R0011</partinfo>||8.6
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-
|-
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|<partinfo>E0840</partinfo>||12.1
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|-
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|<partinfo>J06702</partinfo>||14.7
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|}
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The concentration of all samples was very week. Probably our shaking incubation was week.
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</div>
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-
<!---->
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<div class="culture lysis">
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====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====
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</div>
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-
 
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-
 
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===Friday, July 23 <span class="by">By: Wataru, Tomo, Makoto</span>===
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<div class="miniprep lysis">
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====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
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{| class="experiments"
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!Name||Concentration(ng/&micro;l)
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-
|-
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|<partinfo>pSB4K5</partinfo>||79.2
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-
|-
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|<partinfo>B0015</partinfo>||-
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|}
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We lost <partinfo>B0015</partinfo> by our mistake. The concentration of <partinfo>pSB4K5</partinfo> is high, so this condition of shaking incubation is moderate.
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-
</div>
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<!---->
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<div class="pcr-purification lysis">
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====Picking up number 1, 3, 5, and 7 of the products of PCR, and PCR-purification====
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{| class="experiments"
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-
!No.||Name||Concentration (ng/&micro;l)||New Name
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-
|-
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|1||S-R-Rz/Rz1||18.6||-
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|-
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|3||S||77.6||S<sub>1</sub>
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|-
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|5||S-R-Rz/Rz1||33.6||-
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-
|-
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|7||S||65.4||S<sub>2</sub>
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-
|}
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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">
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====Retry of [[Team:Kyoto/Protocols#Standard_PCR|Standard PCR]] for S-R-Rz/Rz1====
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-
{| class="experiments"
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-
!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
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-
|-
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|1||28&micro;l||3||5||5||5||1.5||1.5||1||50
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|-
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|2||28||3||5||5||5||1.5||1.5||1||50
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-
|-
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|3||26.5||4.5||5||5||5||1.5||1.5||1||50
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-
|-
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|4||26.5||4.5||5||5||5||1.5||1.5||1||50
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-
|-
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|5||25||6||5||5||5||1.5||1.5||1||50
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|-
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|6||25||6||5||5||5||1.5||1.5||1||50
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-
|}
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{|class="experiments"
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-
|+ PCR condition
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-
|-
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|94&#x2103;||2min||
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-
|-
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|98&#x2103;||10sec||rowspan="3"|30 cycles
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-
|-
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-
|55&#x2103;||30sec
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-
|-
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-
|68&#x2103;||4min
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-
|-
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-
|4&#x2103;||forever||
+
-
|}
+
-
</div>
+
-
<!---->
+
-
<div class="digestion">
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-
====[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]] of <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, and PstI to check function of our Restriction Enzyme====
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-
{| class="experiments"
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-
!No.||10xBuffer||BSA||Enzyme||MilliQ||Total||Incubation
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-
|-
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|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
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-
|-
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|2||5||1||''Xba''I 0.1||3.6||10
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-
|-
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|3||5||1||''Spe''I 0.1||3.6||10
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|-
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|4||5||1||''Pst''I 0.1||3.6||10
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|-
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|5||5||1||-||3.7||10
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-
|}
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</div>
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<!---->
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<div class="electrophoresis">
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====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] of above sample for 35min====
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-
[[Image:KyotoExp100723-1.png|300px|right]]
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-
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.
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-
</div>
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-
<!---->
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<div class="digestion lysis">
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====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>====
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{| class="experiments"
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-
!Name||Sample Volume (&micro;l)||10×Buffer||Enzyme 1||Enzyme 2||MilliQ||Total||Incubation
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-
|-
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|S<sub>1</sub>||11||5||''EcoR''I 0.2||''Spe''I 0.2||33.6||50||rowspan="3"|At 37&#x2103; for 2h
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-
|-
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|S<sub>2</sub>||11||5||''EcoR''I 0.2||''Spe''I 0.2||33.6||50
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-
|-
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|<partinfo>E0840</partinfo>||45||5||''EcoR''I 0.2||''Xba''I 0.2||0||50
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-
|}
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After PCR purification, evaporated them and diluted 3ul.
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</div>
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-
<!---->
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<div class="ligation lysis">
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====Ligation====
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{| class="experiments"
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!Name||Vector||Insert||Ligation High||Total
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-
|-
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|S-E0840<sub>1</sub>||<partinfo>E0840</partinfo> 0.5&micro;l||S<sub>1</sub> 0.5||1||2
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-
|-
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|S-E0840<sub>2</sub>||<partinfo>E0840</partinfo> 0.5||S<sub>2</sub> 0.5||1||2
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-
|}
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</div>
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-
 
+
-
 
+
-
===Monday, July 26 <span class="by">By: Wataru, Tomonori, Makoto</span>===
+
-
<div class="electrophorersis lysis">
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====[[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>
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-
<!---->
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-
<div class="pcr-purification">
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====PCR Purification====
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{| class="experiments"
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!No.||Concentration (ng/&micro;l)||New Name
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-
|-
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|4||51.6||SRRz<sub>1</sub>
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-
|-
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|5||59.3||
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|-
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|6||59.6||SRRz<sub>2</sub>
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-
|}
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</div>
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<!---->
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<div class="transformation lysis">
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====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
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-
{| class="experiments"
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-
!Name||Well||Sample (&micro;l)||Competent Cell (&micro;l)||Total (&micro;l)||Plate||Incubation||Result
+
-
|-
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|<partinfo>E0240</partinfo>||1-12-M||1||20||21||LB (Amplicillin+)||rowspan="3"|At 37&#x2103; 7/26 - 7/27||×
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-
|-
+
-
|<partinfo>I20260</partinfo>||2-17-F||1||20||21||rowspan="2"|LB (Kanamycin+)||×
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-
|-
+
-
|<partinfo>J04450</partinfo>||1-5-E||1||20||21||×
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-
|}
+
-
</div>
+
-
<!---->
+
-
<div class="culture lysis measure">
+
-
====Culture of <partinfo>pSB4K5</partinfo>, <partinfo>E0840</partinfo>, and <partinfo>B0015</partinfo>====
+
-
</div>
+
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===Tuesday, July 27 <span class="by">By: Wataru, Tomo, Kazuya, Ken, Naoi===
+
-
<div class="colony-pcr lysis">
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====[[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
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-
|-
+
-
|<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">
+
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====[[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 Copy]||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 Copy]||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 Copy]||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 Copy]||-||1||20||21||rowspan="3"|LB kan||rowspan="3"|8/3~8/4
+
-
|-
+
-
|R0011-E0240<sub>2</sub>[Low Copy]||-||1||20||21
+
-
|-
+
-
|J23101-E0240[Low Copy]||-||1||20||21
+
-
|}
+
-
</div>
+
-
</div>
+

Latest revision as of 11:33, 27 October 2010

Contents

Notebook

Notebooks

^Top

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.

^Top

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