Chiba/Lab Notebook
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== AHL TEAM == | == AHL TEAM == | ||
+ | Fukutomi/Tamaki/Kazama | ||
+ | |||
+ | *Let's make AHL Sender and Receiver | ||
+ | |||
+ | *aiiA Receiver(under constant promoter) | ||
+ | |||
+ | *aiiA Receiver(under lux promter) | ||
+ | |||
+ | *AHL test(diffusion and concentration gradient) | ||
+ | |||
+ | *AHL test on LB plus methionine plate | ||
+ | |||
+ | *Sensitive Receiver(LuxR mutant) | ||
+ | |||
+ | *Super Sender(metK+LuxI) | ||
== FliC TEAM == | == FliC TEAM == | ||
+ | Lajiwara/Guishiken/Tominaga | ||
*pUC19-FliC-Hisの作製 | *pUC19-FliC-Hisの作製 | ||
**LINKER LIGATION | **LINKER LIGATION | ||
Line 7: | Line 23: | ||
##Check Size '''done''' | ##Check Size '''done''' | ||
##Digestion(EcoRI,NcoI) '''done''' | ##Digestion(EcoRI,NcoI) '''done''' | ||
- | ##Zymo Clean( | + | ##Zymo Clean(40μ)→SAP(50μ系)→Zymo Clean(30μ) '''done''' |
#Histag,Histag-antiをアニール '''done''' | #Histag,Histag-antiをアニール '''done''' | ||
##Digestion(EcoRI,NcoI) '''done''' | ##Digestion(EcoRI,NcoI) '''done''' | ||
Line 20: | Line 36: | ||
#1,2をLigation '''done''' | #1,2をLigation '''done''' | ||
#Transformation '''done''' | #Transformation '''done''' | ||
- | #Miniprep | + | #Miniprep '''done''' |
#Digestion test | #Digestion test | ||
*Check Protein | *Check Protein | ||
Line 26: | Line 42: | ||
#Western-Bloting | #Western-Bloting | ||
#Magnetic Beans | #Magnetic Beans | ||
+ | *Toxicity Test | ||
+ | *伝達事項~9/11にすること | ||
+ | #タッパーを買う×10くらい | ||
+ | #下の研究室から金属イオンをもらう | ||
+ | #Swamの結果を写真に撮る→Swam培地は壊れやすいので注意←タッパーを買った後のほうがいいかも | ||
+ | #M9培養のODを測る。0.2~0.4でIPTGを2μ(←たぶん)加える→'''''既に加えました''''' | ||
+ | #M9培養のODが2,0を超えたら集菌→冷凍保存(おそらく午前中に集菌できると思います) | ||
+ | #SDS、Western-Blotting | ||
+ | #Toxicity test用のSwam培地を作る→金属イオン濃度を桁で振ること⇒Swamテストと同様に写真に撮る | ||
+ | *[[chiba/Weekly|Weekly]] |
Latest revision as of 05:13, 12 September 2007
AHL TEAM
Fukutomi/Tamaki/Kazama
- Let's make AHL Sender and Receiver
- aiiA Receiver(under constant promoter)
- aiiA Receiver(under lux promter)
- AHL test(diffusion and concentration gradient)
- AHL test on LB plus methionine plate
- Sensitive Receiver(LuxR mutant)
- Super Sender(metK+LuxI)
FliC TEAM
Lajiwara/Guishiken/Tominaga
- pUC19-FliC-Hisの作製
- LINKER LIGATION
- pUC19-FliCをInverse PCR(五種類のF-Primer,R-Primer→15通りのPCR)⇒vectorの作成 done
- Check Size done
- Digestion(EcoRI,NcoI) done
- Zymo Clean(40μ)→SAP(50μ系)→Zymo Clean(30μ) done
- Histag,Histag-antiをアニール done
- Digestion(EcoRI,NcoI) done
- Ligation done
- Transformation done
- miniprep done
- Digestion test done
- Sequence→FliCに6×His配列が挿入されていることを確認 done
- RBSに問題有り→FliC領域の上流に開始コドンが二つ(RBSが結合する確率が高い)
- 1のvectorにRBSを取り付ける done
- 2-1産物をDigestion(HindⅢ,ApaⅠ) done
- 1,2をLigation done
- Transformation done
- Miniprep done
- Digestion test
- Check Protein
- SDS-PAGE
- Western-Bloting
- Magnetic Beans
- Toxicity Test
- 伝達事項~9/11にすること
- タッパーを買う×10くらい
- 下の研究室から金属イオンをもらう
- Swamの結果を写真に撮る→Swam培地は壊れやすいので注意←タッパーを買った後のほうがいいかも
- M9培養のODを測る。0.2~0.4でIPTGを2μ(←たぶん)加える→既に加えました
- M9培養のODが2,0を超えたら集菌→冷凍保存(おそらく午前中に集菌できると思います)
- SDS、Western-Blotting
- Toxicity test用のSwam培地を作る→金属イオン濃度を桁で振ること⇒Swamテストと同様に写真に撮る