Team:Kyoto/Notebook

From 2010.igem.org

(Difference between revisions)
(→Retry Transforamtion of iGEM Parts)
(→PCR for S-R-Rz/Rz1 and S)
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Dilute λDNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template λDNA was 5ng/µl.
Dilute λDNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template λDNA 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
+
!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
 +
|-
|1||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
|1||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 +
|-
|2||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
|3||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 +
|-
|4||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
|5||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 +
|-
|6||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
|7||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 +
|-
|8||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
|8||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
|}
|}

Revision as of 04:27, 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-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℃2minx1
98℃10secx30 cycle
55℃30sec
68℃1min
4℃forever
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