Team:Kyoto/Notebook

From 2010.igem.org

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{{:Team:Kyoto/Header}}
{{:Team:Kyoto/Header}}
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== Index ==
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==Index==
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== Notebook ==
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==Notebook==
===001: Preparation of iGEM Parts===
===001: Preparation of iGEM Parts===
* By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
* By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
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====Make LB plate====
====Make LB plate====
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Make plates for LB (Ampicillin+) and LB (Kanamycin+).
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* Make plates for LB (Ampicillin+) and LB (Kanamycin+).
====Transformation of iGEM Parts====
====Transformation of iGEM Parts====
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====Discussion 001====
====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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* 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".
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And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance.
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===002: Retry Transformation and Miniprep, PCR===
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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
* By: Wataru, Ken, Makoto, Takuya Yamamoto
* Date: 07/21
* Date: 07/21
* Category: Culture, MasterPlate, Transformation, Parts, PCR, LysisCassette
* Category: Culture, MasterPlate, Transformation, Parts, PCR, LysisCassette
* Protocol: [[Team:Kyoto/Protocol#Use_BioBrick_Parts|Use BioBrick Parts]], [[Team:Kyoto/Protocol#PCR|PCR]]
* 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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|}
 
====Make a Master Plate====
====Make a Master Plate====
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====Retry Transforamtion of iGEM Parts====
====Retry Transforamtion of iGEM Parts====
{|
{|
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|Name||Well<sup>*1</sup>||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result|
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|Name||Well<sup>*1</sup>||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
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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||○
|<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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====PCR for S-R-Rz/Rz1 and S====
====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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* Dilute &lambda;DNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template &lambda;DNA was 5ng/µl.
{|
{|
!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.
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* 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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PCR condition:
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----
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{|
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|94&#x2103;||2min||
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|98&#x2103;||10sec||rowspan="3"|x30 cycle
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|55&#x2103;||30sec
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|68&#x2103;||1min
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|-
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|4&#x2103;||forever||
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Revision as of 04:41, 25 August 2010

Contents

Index

Notebook

001: Preparation of iGEM Parts

  • By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
  • Date: 07/20
  • Category: Antibiotic, Parts, Transformation
  • Protocol: Use BioBrick Parts

Solubilization fo antibiotics

  • 1. Make two mixtures as the following.
    • 1g Ampicillin and 20ml MilliQ (Final concentration 50mg/ml).
    • 0.5g Ampicillin and 10ml MilliQ (Final concentration 50mg/ml).
  • 2. Dispense 1.1ml to 1.5ml tubes.
  • 3. Keep in -20℃ Freezer.

Make LB plate

  • Make plates for LB (Ampicillin+) and LB (Kanamycin+).

Transformation of 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 001

  • 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".

002: Retry Transformation and Miniprep, PCR

  • By: Wataru, Ken, Makoto, Takuya Yamamoto
  • Date: 07/21
  • Category: Culture, MasterPlate, Transformation, Parts, PCR, LysisCassette
  • Protocol: Use BioBrick Parts, PCR

Make a Master Plate

Retry Transforamtion 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 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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