Team:Kyoto/Notebook

From 2010.igem.org

(Difference between revisions)
(→Make LB plate)
Line 49: Line 49:
----
----
 +
 +
===002: ===
 +
* By: Wataru, Ken, Makoto, Takuya Yamamoto
 +
* Date: 07/21
 +
* Category: Culture, MasterPlate, Transformation, Parts, PCR, LysisCassette
 +
* Protocol: [[Team:Kyoto/Protocol#Use_BioBrick_Parts|Use BioBrick Parts]], [[Team:Kyoto/Protocol#PCR|PCR]]
 +
 +
====Result of 001====
 +
{|
 +
!Name||Colony||Next Step
 +
|-
 +
|<partinfo>J23100</partinfo>||Many||rowspan="6"|[[#Make_a_Master_Plate|Make a Master Plate]], [[#Miniprep_of_iGEM_Parts_(Ampicillin_Resistance)|Miniprep]]
 +
|-
 +
|<partinfo>J23105</partinfo>||Many
 +
|-
 +
|<partinfo>J23116</partinfo>||Many
 +
|-
 +
|<partinfo>R0011</partinfo>||Many
 +
|-
 +
|<partinfo>E0840</partinfo>||Many
 +
|-
 +
|<partinfo>J06702</partinfo>||Many
 +
|-
 +
|<partinfo>pSB4K5</partinfo>||No||rowspan="2"|[[#Retry_Transformation of iGEM Parts|Retry Transformation]]
 +
|-
 +
|<partinfo>B0015</partinfo>||No
 +
|}
 +
 +
====Make a Master Plate====
 +
 +
====Retry Transforamtion of iGEM Parts====
 +
{|
 +
|Name||Well<sup>*1</sup>||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result|
 +
|-
 +
|<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>B0015</partinfo>||1-23-L||1||20||21||LB (Kanamycin+)||○
 +
|}
 +
* *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 &lambda;DNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template &lambda;DNA was 5ng/µl.
 +
{|
 +
!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
 +
|1||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 +
|2||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 +
|3||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 +
|4||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 +
|5||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 +
|6||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 +
|7||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 +
|8||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 +
|}
 +
* Forward Primer of S-R-Rz/Rz1 and S is common.
 +
{|
 +
|+ PCR condition
 +
|-
 +
|94&#x2103;||2min||x1
 +
|-
 +
|98&#x2103;||10sec||rowspan="3"|x30 cycle
 +
|-
 +
|55&#x2103;||30sec
 +
|-
 +
|68&#x2103;||1min
 +
|-
 +
|4&#x2103;||forever
 +
|}

Revision as of 04:24, 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:

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

Result of 001

NameColonyNext Step
<partinfo>J23100</partinfo>ManyMake a Master Plate, Miniprep
<partinfo>J23105</partinfo>Many
<partinfo>J23116</partinfo>Many
<partinfo>R0011</partinfo>Many
<partinfo>E0840</partinfo>Many
<partinfo>J06702</partinfo>Many
<partinfo>pSB4K5</partinfo>NoRetry Transformation
<partinfo>B0015</partinfo>No

Make a Master Plate

Retry Transforamtion of iGEM Parts

NameWell*1Sample (µl)Competent Cells (µl)Total (µl)PlateIncubation
<partinfo>pSB4K5</partinfo>1-5-G12021LB (Kanamycin+)At 37℃ 7/21 20:50 - 7/22 16:30○
<partinfo>B0015</partinfo>1-23-L12021LB (Kanamycin+)○
  • *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.

NumberWater25mM 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℃2minx1
98℃10secx30 cycle
55℃30sec
68℃1min
4℃forever
Retrieved from "http://2010.igem.org/Team:Kyoto/Notebook"