IGEM:Paris Bettencourt 2012/Notebooks/Semantic group/day by day//2012/07/22: Difference between revisions

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(Autocreate 2012/07/22 Entry for IGEM:Paris_Bettencourt_2012/Notebooks/Semantic_group/day_by_day/)
 
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==Entry title==
==Result Ligation==
* Insert content here...


{| border = "1" width="45%"
! width="35%" | Plate
! width="5%" | colonies?
|-
|Ligation pur
||+++
|-
|Ligation 1/10
|~280
|-
|positive transformation control (pSB1A2::R0011)
||+++
|-
|negative ligation control (pSC002 dig. ie. insert)
|0
|-
|negative ligation control (pSC003 dig. ie. vector)
|~240
|-
|negative transformation control (NEB only)
|0
|-
|}
There is a background of self ligation, I expect that 1 over 10 bacteria have the wrong plasmid.
So I make a PCR colony, and I make a culture of colonies I PCR.
* Tubes L1->L12 are clones from ligation plates.
* Tubes T-1->T-3 are clones from negative control plate (vector)
Here is the content of 1 tube of PCR :
* VR (diluted 10 times from the stock tube) : 2,5µL
* VF2 (diluted 10 times from the stock tube) : 2,5µL
* PCR MM 2X : 25 µL
* water : 20 µL
Here is the PCR pgm :
* 95°C - 15' (to lyse cells too)
* 95°C - 30"
* 50°C - 30"
* 72°C - 40"
* 72°C - 10'
* 4°C - <math>\infty</math>


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__NOTOC__
__NOTOC__
[[category:OWWLabNotebookV1]]
[[category:OWWLabNotebookV1]]

Revision as of 07:12, 22 July 2012

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Result Ligation

Plate colonies?
Ligation pur +++
Ligation 1/10 ~280
positive transformation control (pSB1A2::R0011) +++
negative ligation control (pSC002 dig. ie. insert) 0
negative ligation control (pSC003 dig. ie. vector) ~240
negative transformation control (NEB only) 0

There is a background of self ligation, I expect that 1 over 10 bacteria have the wrong plasmid. So I make a PCR colony, and I make a culture of colonies I PCR.

  • Tubes L1->L12 are clones from ligation plates.
  • Tubes T-1->T-3 are clones from negative control plate (vector)

Here is the content of 1 tube of PCR :

  • VR (diluted 10 times from the stock tube) : 2,5µL
  • VF2 (diluted 10 times from the stock tube) : 2,5µL
  • PCR MM 2X : 25 µL
  • water : 20 µL

Here is the PCR pgm :

  • 95°C - 15' (to lyse cells too)
  • 95°C - 30"
  • 50°C - 30"
  • 72°C - 40"
  • 72°C - 10'
  • 4°C - [math]\displaystyle{ \infty }[/math]
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