Team:Kyoto/Notebook

From 2012.igem.org

(Difference between revisions)
(August 2)
(August 2)
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* [[Team:Kyoto/Notebook1|Notebook1]]: Florigen
* [[Team:Kyoto/Notebook1|Notebook1]]: Florigen
==August 2==
==August 2==
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'''Mutation of FT'''
 
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{|class="wikitable"
 
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|+Inverse PCR
 
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!10xBufer!!2mM dNTPs!!primer fwd!!primer rev!!template!!polymerase!!MilliQ!!Total
 
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|-
 
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|5||5||1.5||1.5||0.5||1||35.5||50
 
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|}
 
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94°C 2min, (98°C 10sec, 68°C 4min)x2cycles, 4°C Hold
 
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{|class="wikitable"
 
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|+Dpn1 Digestion
 
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!PCR product!!Dpn1
 
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|-
 
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|50||2
 
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|}  37℃,1h incubate
 
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{|class="wikitable"
 
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|+Self-ligation
 
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!product!!MilliQ!!Ligase!!T4 Kinase!!Total
 
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|-
 
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|2||7||5||1||15
 
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|}
 
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16°C, 1h incubate
 
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{|class="wikitable"
 
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|+Transformation
 
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!competent cell!!DNA
 
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|-
 
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|20||2
 
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|}
 
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Cells were stored on ice for 30min. <br>
 
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After 42°C 60sec heat shock, cells were stored on ice for 2min.<br>
 
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Then cells were preincubated at 37°C for 1hr, plated to Kanamycin plate.
 
==August 10==
==August 10==

Revision as of 13:13, 21 August 2012

August 2

August 10

Golden Gate Assembly method This method helps us to constract some genes quickly and we can design the order of constractions. We are trying to construct a gene cycle composed of 6 genes( pSB1A3, lacI, GFP, RFP, GFP, DT). After this experiment is confirmed to work, we will so some experiment to check how the numbers of promorters influences the strength of transcription.


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