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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[[#top-section|^Top]]
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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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===Other Information===
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|-
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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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|colspan="2"|Dispense 1.1ml of the solution into 1.5ml tubes||
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* [[Team:Kyoto/Materials|Materials]]: Strains, DNA, and Primers.
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|-
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* [[Team:Kyoto/Parts|Parts]]: Construction of each part and BioBrick Parts used in our project.
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|colspan="2"|Store in the freezer (-20&#x2103;)||
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|}
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[[#top-section|^Top]]
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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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===Wednesday, July 21 <span class="by">By: Wataru, Ken, Makoto, Takuya Y.</span>===
 
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<div class="culture lysis measure">
 
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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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</div>
 
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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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|+ 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||
 
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|}
 
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</div>
 
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===Thursday, July 22 <span class="by">By: Wataru</span>===
 
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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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===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.
 
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</div>
 
-
----
 
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<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||
 
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|}
 
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</div>
 
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----
 
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<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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</div>
 
-
----
 
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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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<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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</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"