Team:Kyoto/Notebook

From 2010.igem.org

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==Notebook==
==Notebook==
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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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{| class="note"
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* 1. Make two mixtures as the following.
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|+Tuesday, July 20
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** 1g Ampicillin and 20ml MilliQ (Final concentration 50mg/ml).
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|-
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** 0.5g Ampicillin and 10ml MilliQ (Final concentration 50mg/ml).
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|By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
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* 2. Dispense 1.1ml to 1.5ml tubes.
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|-
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* 3. Keep in -20℃ Freezer.
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|Category: Antibiotic, Transformation
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|-
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====Make LB plate====
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|[[Team:Kyoto/Protocols#Solubilization_of_Antibiotics|Solubilized of Antibiotics]], Ampicillin (1g) and Kanamycin (0.5g).
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* Make plates for LB (Ampicillin+) and LB (Kanamycin+).
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|-
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|[[Team:Kyoto/Protocols#Making_Plate|Made plates]] for LB (Ampicillin+) and LB (Kanamycin+).
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====Transformation of iGEM Parts====
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|-
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{|
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|[[Team:Kyoto/Protocols#Transformation|Transformed]] iGEM Parts.
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{| class="experiments"
!Name||Well<sup>*1</sup>||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
!Name||Well<sup>*1</sup>||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
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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.
* *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'''
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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+).
* A vector of "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Ampicillin+).
* And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance.
* 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".
* So, it was predicted that we will fail the transformation of "pSB4K5" and "B0015".
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|}
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===002: Retry Transformation and Miniprep, PCR===
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----
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* By: Wataru, Ken, Makoto, Takuya Yamamoto
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{| class="note"
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* Date: 07/21
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|+Wednesday, July 21
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* Category: Culture, MasterPlate, Transformation, Parts, PCR, LysisCassette
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|-
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* Protocol: [[Team:Kyoto/Protocol#Use_BioBrick_Parts|Use BioBrick Parts]], [[Team:Kyoto/Protocol#PCR|PCR]]
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|By: Wataru, Ken, Makoto, Takuya Yamamoto
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|-
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====Culture and Make master plates====
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|Category: Transformation, PCR, Lysis Cassette
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* Cultured plates in which colonies was observed at 37&#x2103; 07/21 20:50 - 07/22 17:00.
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|-
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* Made master plates of the above plates.
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|Cultured plates in which colonies was observed at 37&#x2103; from 07/21 20:50 to 07/22 17:00.
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|-
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====Retry Transforamtion of iGEM Parts====
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|Made a master plate of the above plates.
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{|
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|-
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|Retried Transformation of iGEM Parts.
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{| class="experiments"
|Name||Well<sup>*1</sup>||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
|Name||Well<sup>*1</sup>||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
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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.
* *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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|-
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====PCR for S-R-Rz/Rz1 and S====
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|PCR PCR for S-R-Rz/Rz1 and S
* Dilute &lambda;DNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template &lambda;DNA was 5ng/µl.
* 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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{| class="experiments"
!No.||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
!No.||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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|-
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* Forward Primer of S-R-Rz/Rz1 and S is common.
* Forward Primer of S-R-Rz/Rz1 and S is common.
* PCR condition : 94&#x2103; x 2min, (98&#x2103; x 10sec, 55&#x2103; x 30sec, 68&#x2103; x 1min) x 30cycles, 4&#x2103; forever
* PCR condition : 94&#x2103; x 2min, (98&#x2103; x 10sec, 55&#x2103; x 30sec, 68&#x2103; x 1min) x 30cycles, 4&#x2103; forever
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|}
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Revision as of 02:18, 27 August 2010

Contents

Index

Notebook

Tuesday, July 20
By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
Category: Antibiotic, Transformation
Solubilized of Antibiotics, Ampicillin (1g) and Kanamycin (0.5g).
Made plates for LB (Ampicillin+) and LB (Kanamycin+).
Transformed iGEM Parts.
NameWell*1Sample (µl)Competent Cells (µl)Total (µl)PlateIncubationResult
<partinfo>J23100</partinfo>1-18-C12021LB (Ampicillin+)At 37℃ 7/20 20:50 - 7/21 17:00○
<partinfo>J23105</partinfo>1-18-M12021○
<partinfo>J23116</partinfo>1-20-M12021○
<partinfo>R0011</partinfo>1-6-G12021○
<partinfo>E0840</partinfo>1-12-O12021○
<partinfo>J06702</partinfo>2-8-E12021○
<partinfo>pSB4K5</partinfo>1-5-G12021×
<partinfo>B0015</partinfo>1-23-L12021LB (Kanamycin+)×
  • *1 "1-18-C" means well 18C in [http://partsregistry.org/Help:Spring_2010_DNA_distribution Spring 2010 DNA Distribution Kit] Plate 1.
  • Discussion
  • A vector of "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Ampicillin+).
  • 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".
Wednesday, July 21
By: Wataru, Ken, Makoto, Takuya Yamamoto
Category: Transformation, PCR, Lysis Cassette
Cultured plates in which colonies was observed at 37℃ from 07/21 20:50 to 07/22 17:00.
Made a master plate of the above plates.
Retried Transformation of iGEM Parts.
NameWell*1Sample (µl)Competent Cells (µl)Total (µl)PlateIncubationResult
<partinfo>pSB4K5</partinfo>1-5-G12021LB (Kanamycin+)At 37℃ 7/21 20:50 - 7/22 16:30○
<partinfo>B0015</partinfo>1-23-L12021○
  • *1 "1-5-G" means well 5G in [http://partsregistry.org/Help:Spring_2010_DNA_distribution Spring 2010 DNA Distribution Kit] Plate 1.
PCR PCR for S-R-Rz/Rz1 and S
  • Dilute λDNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template λDNA was 5ng/µl.
No.Water25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2TemplateDNA (5ng/µl)Primer S-R-Rz/Rz1 Forward (10µM)Primer S-R-Rz/Rz1 Reverse (10µM)Primer S Reverse (10µM)KOD Plus ver.2Total
128µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
228µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
328µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
428µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
528µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
628µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
728µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
828µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
  • Forward Primer of S-R-Rz/Rz1 and S is common.
  • PCR condition : 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever
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