Team:Kyoto/Notebook

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{{:Team:Kyoto/Header}}
{{:Team:Kyoto/Header}}
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== Index ==
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==Notebook==
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===Notebooks===
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* [[Team:Kyoto/Notebook1|Notebook1]]: Construction for Lysisbox.
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* [[Team:Kyoto/Notebook2|Notebook2]]: Measurement of R0011.
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* [[Team:Kyoto/Notebook3|Notebook3]]: Measurement of Lysis Cassette and Lysisbox etc.
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== Notebook ==
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[[#top-section|^Top]]
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===001: Preparation of iGEM Parts===
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* By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
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* Date: 07/20
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* Category: Antibiotic, Parts, Transformation
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* Protocol: [[Team:Kyoto/Protocol#Use_BioBrick_Parts|Use BioBrick Parts]]
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====Solubilization fo antibiotics====
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===Other Information===
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* 1. Make two mixtures as the following.
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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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** 1g Ampicillin and 20ml MilliQ (Final concentration 50mg/ml).
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* [[Team:Kyoto/Materials|Materials]]: Strains, DNA, and Primers.
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** 0.5g Ampicillin and 10ml MilliQ (Final concentration 50mg/ml).
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* [[Team:Kyoto/Parts|Parts]]: Construction of each part and BioBrick Parts used in our project.
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* 2. Dispense 1.1ml to 1.5ml tubes.
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* 3. Keep in -20℃ Freezer.
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====Make LB plate====
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[[#top-section|^Top]]
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Make plates for LB (Ampicillin+) and LB (Kanamycin+).
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====Transformation of iGEM Parts====
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{|
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!Name||Well<sup>*1</sup>||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
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|-
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|<partinfo>J23100</partinfo>||1-18-C||1||20||21||rowspan="7"|LB (Ampicillin+)||rowspan="8"|At 37℃ 7/20 20:50 - 7/21 17:00||○
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|-
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|<partinfo>J23105</partinfo>||1-18-M||1||20||21||○
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|-
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|<partinfo>J23116</partinfo>||1-20-M||1||20||21||○
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|-
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|<partinfo>R0011</partinfo>||1-6-G||1||20||21||○
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|-
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|<partinfo>E0840</partinfo>||1-12-O||1||20||21||○
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|-
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|<partinfo>J06702</partinfo>||2-8-E||1||20||21||○
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|-
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|<partinfo>pSB4K5</partinfo>||1-5-G||1||20||21||×
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|-
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|<partinfo>B0015</partinfo>||1-23-L||1||20||21||LB (Kanamycin+)||×
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|}
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* *1 "1-18-C" means well 18C in [http://partsregistry.org/Help:Spring_2010_DNA_distribution Spring 2010 DNA Distribution Kit] Plate 1.
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====Discussion 001====
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A vector of "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Ampicillin+).
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And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance.
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So, it was predicted that we will fail the transformation of "pSB4K5" and "B0015".
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----
----
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===002: ===
 
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* By: Wataru, Ken, Makoto, Takuya Yamamoto
 
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* Date: 07/21
 
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* Category: Culture, MasterPlate, Transformation, Parts, PCR, LysisCassette
 
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* Protocol: [[Team:Kyoto/Protocol#Use_BioBrick_Parts|Use BioBrick Parts]], [[Team:Kyoto/Protocol#PCR|PCR]]
 
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====Result of 001====
 
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{|
 
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!Name||Colony||Next Step
 
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|-
 
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|<partinfo>J23100</partinfo>||Many||rowspan="6"|[[#Make_a_Master_Plate|Make a Master Plate]], [[#Miniprep_of_iGEM_Parts_(Ampicillin_Resistance)|Miniprep]]
 
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|-
 
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|<partinfo>J23105</partinfo>||Many
 
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|-
 
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|<partinfo>J23116</partinfo>||Many
 
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|-
 
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|<partinfo>R0011</partinfo>||Many
 
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|-
 
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|<partinfo>E0840</partinfo>||Many
 
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|-
 
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|<partinfo>J06702</partinfo>||Many
 
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|-
 
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|<partinfo>pSB4K5</partinfo>||No||rowspan="2"|[[#Retry_Transformation of iGEM Parts|Retry Transformation]]
 
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|-
 
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|<partinfo>B0015</partinfo>||No
 
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|}
 
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====Make a Master Plate====
 
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====Retry Transforamtion of iGEM Parts====
 
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{|
 
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|Name||Well<sup>*1</sup>||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 (Kanamycin+)||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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* *1 "1-5-G" means well 5G in [http://partsregistry.org/Help:Spring_2010_DNA_distribution Spring 2010 DNA Distribution Kit] Plate 1.
 
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====PCR for S-R-Rz/Rz1 and S====
 
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Dilute &lambda;DNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template &lambda;DNA was 5ng/µl.
 
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{|
 
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!Number||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||TemplateDNA (5ng/µl)||Primer S-R-Rz/Rz1 Forward (10µM)||Primer S-R-Rz/Rz1 Reverse (10µM)||Primer S Reverse (10µM)||KOD Plus ver.2||Total
 
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|1||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 
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|2||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 
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|3||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 
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|4||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 
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|5||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 
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|6||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 
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|7||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 
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|8||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 
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|}
 
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* Forward Primer of S-R-Rz/Rz1 and S is common.
 
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{|
 
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|+ PCR condition
 
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|-
 
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|94&#x2103;||2min||x1
 
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|-
 
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|98&#x2103;||10sec||rowspan="3"|x30 cycle
 
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|-
 
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|55&#x2103;||30sec
 
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|-
 
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|68&#x2103;||1min
 
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|-
 
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|4&#x2103;||forever
 
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|}
 

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"