August/5 August 2009

From 2009.igem.org

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   BBa_C0078 (mainly from  "signal senders"and "signal receivers")
   BBa_C0078 (mainly from  "signal senders"and "signal receivers")
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<br>
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5th.August<br>
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wet work<br>
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1.'''Mini prep'''<br>
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5th.August
 
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wet work
 
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1.Mini prep
 
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  Culture broth (5ml) was centrifuged (15K,1min, room temperature) to harvest the cells.
  Culture broth (5ml) was centrifuged (15K,1min, room temperature) to harvest the cells.
  The cells ware suspended in 250μl of P1　for cell lysis and cutting RNA. 250μl of P2 was added,  
  The cells ware suspended in 250μl of P1　for cell lysis and cutting RNA. 250μl of P2 was added,  
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  those suspension ware on ice for 3min. They ware centrifuged (15K,10min,4℃).
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  those suspension ware on ice for 3min. They ware centrifuged (15K,10min,4°C).
  Those supernatants ware applied to blue columns.Then they ware centrifuged(13K,1min,room temperature)  
  Those supernatants ware applied to blue columns.Then they ware centrifuged(13K,1min,room temperature)  
  and those solution ware applied and centrifuged (13K,1min,room temperature)again to collect those DNAs.
  and those solution ware applied and centrifuged (13K,1min,room temperature)again to collect those DNAs.
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  Those resulting liquids ware tested with Nano Drop.
  Those resulting liquids ware tested with Nano Drop.
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※the way to measure using Nano Drop is not so difficult,so It is skipped.
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Result<br>
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Result
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  plate2 8-E  78.6ng/μl
  plate2 8-E  78.6ng/μl
   
   
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2.Transformation
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2.'''Transformation'''<br>
   
   
  plate3 18-0 (BBa_K143032) was transformed.
  plate3 18-0 (BBa_K143032) was transformed.
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<br>
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3.'''Cutting with restriction enzyme'''<br>
   
   
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The way is skipped because it was noted before.
 
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3.Cutting with restriction enzyme
 
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ちょっと挫折したんで日本語いれつつ。
 
  About mole, inserted part is needed from three to six times as much as vector.
  About mole, inserted part is needed from three to six times as much as vector.
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  20μlになるように水、酵素、ベクター、挿入部分をもつもの(今回はこれもベクター)の組成を考えて混ぜる。(酵素は0.5~1.0μl)
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  20uL (dH2O , enzyme , Vector , Insert) (enzyme : 0.5~1.0 ul)
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  37℃でインキュベート3時間。
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  37°C , 3hr
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最後適当感ありますが、横からみて勉強してただけなので。
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[https://2009.igem.org/Team:Osaka/NOTES back to NOTES]
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英語変なとこあったら直しおねがいします。一応論文の英語を参考にしたんですが・・・
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あと提案ですが文責はかくようにしましょ。
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written by Rie Takino
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Latest revision as of 07:33, 12 October 2009

　To All 

 From Tadasi

 I checked these following parts today.

  BBa_C0062 BBa_F2620 BBa_F2621 BBa_F2622 BBa_C0171 BBa_C0179 BBa_C0079 BBa_F1610 BBa_C0061 BBa_C0161 BBa_C0070
 BBa_C0078 (mainly from  "signal senders"and "signal receivers")


5th.August

wet work

1.Mini prep

Culture broth (5ml) was centrifuged (15K,1min, room temperature) to harvest the cells.
The cells ware suspended in 250μl of P1　for cell lysis and cutting RNA. 250μl of P2 was added, 
and tubes ware turned over 4,5 times.
Then,350μl of N3 was added,and tubes ware turned over 4,5 times.


those suspension ware on ice for 3min. They ware centrifuged (15K,10min,4°C).
Those supernatants ware applied to blue columns.Then they ware centrifuged(13K,1min,room temperature) 
and those solution ware applied and centrifuged (13K,1min,room temperature)again to collect those DNAs.


Those resulting pellets ware suspended in 500μl of buffer PB, and they ware centrifuged(13K,1min,room temperature).
Those resulting pellets ware suspended in 750μl of PE, and they ware centrifuged(13K,1min,room temperature).
Those resulting pekkets ware centrifuged(14K,3min,roomtenperature)again to remove PE.


70μl of buffer EB was applied those columns,and left for 5min.Then,they ware centrifuged(14K,1.5min,room temperature)



Those resulting liquids ware tested with Nano Drop.

Result

plate2 8-E  78.6ng/μl

plate1 8-I  88.0ng/μl

plate1 8-K  91.0ng/μl

plate1 12-M 89.7ng/μl

2.Transformation

plate3 18-0 (BBa_K143032) was transformed.


3.Cutting with restriction enzyme

About mole, inserted part is needed from three to six times as much as vector.
20uL (dH2O , enzyme , Vector , Insert) (enzyme : 0.5~1.0 ul)
37°C , 3hr

back to NOTES

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