Team:Hong Kong HKU/team/people

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Shannon<br><br>
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Samson<br><br>
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Ting-Yen<br><br>
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Lai Hei Ming<br><br>
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Charmaine<br><br>
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Fall<br><br>
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Mickey<br><br>
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Kenny<br><br>
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Calvin<br><br>
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<p><font face="Arial" size="5">Talk delivered to Sec School Students</font></p><br>
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June 28th, Friday
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Long Amp PCR + Cloning
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Cargo Team: (Only KO genome available)
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Native Ko_ppk Amplified > Gel Purified (49ng/ul) > TA vector Ligation+Transformation > Spread Plate
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1st step Signal Ko_ppk Am
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Surface Team:
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plified > Gel Purified (weak) > 2nd PCR
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Native EutS Amplified > Gel Purified (25ug/ul) > TA vector Ligation+Transformation > Spread Plate
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His_EutS Amplified > Gel Purified > 2nd PCR
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Flag_EutS Amplified > Gel Purifed > 2nd PCR
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Ko native ppk and Ko sigppk 1st PCR product.
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June 29th, Saturday
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Long Amp PCR + Cloning, continuous PCR and TA vector transformation.
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June 30th, Sunday
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His Euts TA colonies for culture.
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Redo transformation of native EutS and Flag EutS.
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Continuous PCR of Tf_ppk1 and Ko_ppk1.
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July 1st, Monday
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Miniprep of HisEutS and Ko_ppk1 TA clone culture and Digestion test.
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Overnight ligation of Native Ko_ppk1, Native Tf_ppk1, pLacHisEutS, Sig_Koppk1(Weak), Flag_EutS
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July 2nd, Tuesday
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Mass Digestion of His_EutS.
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Continuous PCR and gel purification.
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Miniprep for EutS Topo conlonies.
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4 Transfromation (Topo Clone) : Native Ko_ppk1, Native Tf_ppk1, pLacHisEutS, Sig_Koppk1(Weak), Flag_EutS
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July 3rd, Wednesday
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Continuous PCR of Ko_sigppk, His_EutSMNLK and Flag_EutSMNLK.
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Mass Digestion of native EutS.
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Transformation: His_Eut into K525998 (Cm Plate); TF_sigppk into Topo (Amp+Xgal Plate); Flag_EutS into Topo (1),(2)
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July 4th, Thursday
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MiniPrep and Digestion Test: Native Ko_ppk1, Native Tf_ppk1, Sig_Koppk1
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Transformation: NativeEutS into 525 (1,2) Cm Plate; Native Tfppk, Koppk, sig_koppk
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Prepare DH10B Competent Cells.
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July 5th, Friday
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Miniprep and Digestion Test: HisEutS 525, FlagEutS topo, Tf_sigppk topo.
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2nd Round PCR for His,Flag EutSMNLK
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July 6th, Saturday; July 7th, Sunday; July 8th, Monday
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A very Tough Weekend (6-8/7)
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PCR Native Ko, Native Tf, Sig Tf and Sig Ko was ligated in to Topo Vector.
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Many White Colonies were observed. (Also with a few Blue Colonies)
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Very HAPPY about the result and picked colonies for culture.
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Culture Two Times and Found out the growth rate was abnormally Slow and the Culture was not cloudy and contain fillament like clusters.
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Maybe the ppk gene might be toxic to cell?<< Wrong
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Maybe the shaker speed and temperature not correct? << Wrong
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WE have Fungus/other bacteria Comtamination!!!
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Hints:
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Colonies morphology>>very white and not transparent and moist compared to Ecoli colonies
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Culture grow well (withou filament clusture) in 32C.
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Can grow in Wrong Antibiotics. (Topo vector is Kana resistant, I use Cm to test>Still grow)
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Tried to miniprep on tube with less clusters. No Plasmid.
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Right Now We don't Know the source of contamination.
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Overview:
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What we have:
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We have EutS, His EutS, Flag EutS in Topo Vector. Positive Digestion Pattern.
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Send for sequencing on 9/7.
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To avoid comtaminantion.
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Will Redo Transformation to create a new stock.
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But Right now, we have:
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HisEutS 1,2 glycerol stock and streak plates.
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FlagEutS 1,2 glycerol stock and streak plates.
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(picked colonies from plates and tested postive, but cannot tell whether there is comtamination)
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EutS Retransformed plates and glycerol stock.
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Previous EutS, His_EutS, Flag_EutS in to 525 vector failed. Will try again.
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But we still don't have any ppk in vector!! But have succeed in PCR (But result random)
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Cannot Amplify EutSMNLK from K311004. Will send K311004 for sequencing.
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July 9th, Tuesday
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Miniprep and Digestion Test of Tf native ppk, Tf sigppk and Ko sigppk.
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Redo ligation and transformation of Ko native ppk, Ko sigppk and Tf sigppk.
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July 10th, Wednesday
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PCR of Ko native ppk, Tf sigppk, Ko sigppk
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Tranformation of 4 kind of ppk1.
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Mass Digestion of Tf native ppk plasmid 1,2
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Overnight culture of Tf native ppk1.
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July 11th, Thursday
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Colony PCR.
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MiniPrep of three Tf native ppk
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Making competent cells.
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Ligation of Tf native DNA (XbaI/ PstI) with 525 vector.
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Transformation of Tf sigppk.
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July 12th, Friday
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1st round PCR of KO sigppk1
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Miniprep of 12 tubes; Digestion test
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July 13th, Saturday
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Digestion test.
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Picked EutS 4, HisEutS 1, FlagEutS 1, Tfppk 2 plasmid transformed into BL21
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Colony PCR of sigppk.
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July 14th, Sunday
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Check plates (Ko native ppk and Ko sigppk 1st PCR product) → pick 34 colonies for colonies PCR → Run gel, two from ko native ppk showed 2100bp. → Culture these two colonies in 5ml LB overnight.
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Digestion of Tf sigppk plasmid (XbaI and PstI) and purify 2100bp tf sigpkk DNA → Overnight ligation with 525 vector.
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Ligation of Ko sigppk PCR product into topo vector → Trasformation
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July 15th, Monday
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Miniprep and Digestion Test: Ko native ppk.
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Transformation of Tf sigppk 525 vector (into Cm plate), Ko sigppk topo vector (into Kan plate)
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Pick two colonies from Tf sigppk stock plate and culture overnight.
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July 16th, Tuesday
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Check plates (Tf sigppk 525, Ko sigppk topo) and colonies PCR, culture 6 colonies.
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Digestion  Ko native ppk overnight.
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July 17th, Wednesday
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Miniprep and digestion test of Topo Ko sig 1,2,3 and Tf sigppk 525 1,2,3.
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Digestion overnight (Ko sigppk 3, Tf sigppk1 and K525998 plasmids)
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Gel Purification of digestion product.
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July 18th, Thursday
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Run gel and purification of Digestion Product.
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Ligation of Ko native ppk and Tf sigppk to new 525 vector, transformation.
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Transformation of Ko native ppk/525 into DH10B, spread plate.
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Pick two colonies from Ko native ppk and one colony from Ko sigppk stock plates and culture overnight.
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July 19th, Friday
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Colonies PCR of Ko native ppk 525, Tf sigppk 525.
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MiniPrep of yesterday's culture. ( Ko native ppk topo and Ko sigppk topo)
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Pick 2 colonies from Euts native525, 2 from Flag Euts525, 2 from His Euts525, 1 from Tf native ppk525 and culture overnight.
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July 20th, Saturday
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Antibiotics Mistake, all culture failed. Redo culture again.
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July 21st, Sunday
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MiniPrep and digestion test of Ko native ppk 525, Tf sigppk525.
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Transformation of Ko native ppk 525 vector into Bl21 and spread plates.
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Culture Tf native ppk 525 BL21 overnight. Culture 4 tubes of K311004 DH10B overnight.
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July 22nd, Monday
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Check plates: No colonies. Thus redo transformation of Ko native ppk 525 vector into Bl21.
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Miniprep of  K311004 DH10B, concentrate into one plasmid purification kit. Get a high conc. plasmid sample (110.4ng/ul)
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IPTF induction of Tf native ppk Bl21, culture overnight.
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July 23rd, Tuesday
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Run PAGE  of induced Tf native ppk Bl21 cell lysate and control, coomassie blue staining.
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July 24th, Wednesday
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As yesterday's result was not ideal, redo PAGE and coomassie blue staining.
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July 25th, Thursday
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Check the coomassie blue staining result: A 500kDa band (referring to the sequencing result, it might be a premature stop codon. normal ppk: 78kDa)
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PCR and Purification: Ko native ppk(Template: Ko genomic DNA); Tf sigppk1 2nd PCR(template: WY's Tf sigppk1 1st PCR product); Ko sigppk 1st PCR (template: new making Ko native ppk)
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PCR test: plasmid Sigppk1 topo 1, Plasmid sigppk1 topo 2
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Culture Ko sigppk topo 1,2,3 overnight.
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Culture Tf native ppk bl21 ovenight.
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July 26th, Friday
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IPTG inductionof Tf native ppk bl21(10, 20).
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Miniprep and Digestion test of Ko sigppk topo 1,2,3.
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Run gel of Ko sigppk 3 PCR product.
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Ligation of Tf sigppk and Ko native ppk into topo vector and transformation
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Culture Euts native, Flag, His Bl21 overnight.
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July 27th, Saturday
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Run PAGE of Tf native ppk bl21, coomassie blue staining.
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IPTG induction of Euts Native, Flag, His.
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Check plates. (White colonies, Contamination?)
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July 28th, Sunday
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Colony PCR of TF sigppk and Ko native ppk topo DH10B
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Run PAGE of Euts Native, Flag, His Bl21. Coomassie Blue staining.
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Transformation of Tf sigppk topo and spread plate.
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July 29th, Monday
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Check plate of Tf sigppk topo, colony PCR.
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Miniprep of Ko native ppk topo and Digestion test.
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IPTG induction of Tf native ppk.
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Transformation of Euts native, flag, his; Tf sigppk 1, 5 (all in 525 vector) into BL21 and spread plates.
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July 30th, Tuesday
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Run gel of digestion product, purification. Ligation into 525 vector.
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Run PAGE of Tf native ppk and coomassie blue staining.
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Miniprep of Tf sigppk topo.
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Culture Euts native, flag, his; Tf sigppk 1, 5 BL21 colonies overnight for IPTG induction.
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July 31st, Wednesday
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IPTG induction of Euts native, flag, his; Tf sigppk 1, 5
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Digestion of Tf sigppk topo and digestion test. Ligation into 525 vector.
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Transformation of Ko native ppk 525 into DH10B and spread plates.
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August 1st, Thursday
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Run PAGE gel of Euts native, flag, his; Tf sigppk 1, 5 and Coomassie blue staining.
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Transformation of Tf sigppk 525 into DH10B and spread plate.
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Colony PCR of Ko native ppk 525 DH10B
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1st Round PCR of Tf sigppk.
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August 2nd, Friday
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Miniprep and digestion test.
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Colony PCR.
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August 3rd, Saturday
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Miniprep of Tf sigppk, Ko native ppk in DH10B ,tf native ppk 5, native EutS, Flag EutS, His EutS in BL21. Digestion test.
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Try IPTG induction again using a new condition.
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Transformation of Tf sigppk 2,5, 7 in BL21.
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August 4th, Sunday
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PAGE of EutS native, Flag EutS and HisEutS.
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August 5th, Monday
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August 6th, Tuesday
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August 7th, Wednesday
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August 8th, Thursday
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August 9th, Friday
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August 10th, Saturday
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PCR ppk for commercial vector(adding tag for purification)
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PCR sig-RFP for detecting localization
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Transformation of KO525 (4, 19) into bl21
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Transformation of ligation product (RFP into 525)
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1st PCR of Sig Ko
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August 11th, Sunday
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Colony PCR of RFP-525 plates, culture positive colonies
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Gel Purify 1st PCR SigKo, 2nd PCR
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Transform Ko525(4), Ko525(19) into DH5a for DNA storage
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Overnight Culture BL21: KO525(4)(19),TFsig(2),TF(6)(7)(8)
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Transform Ko Topo 1, Ko Topo 2, Ko Topo 3, Tf Topo A, Tf topo B into BL21 and  spread plates and grow overnight.
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Second PCR sig-RFP, Purified and digest using S+P overnight
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Digest 525948 Vector using S+P overnight
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Ligatation of sig 2nd PCR product and TOPO
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August 12th, Monday
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Miniprep RFP525 (2,4,5,6)>>Digestion test>>Transform (4) into BL21
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Gel purify 2nd SigKo, SigRFP >> Ligate into T-vector>> Transform DH5a
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Transform Pet28a (kana R) into DH5a to get vector
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Transform K311004,HisSMNLK,FlagSMNLK into BL21
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Streak HisSMNLK (1a),FlagSMNLK(4b) DH5a to get DNA
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Culture NativeEutS, HisS, FlagS 525 to get DNA
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IPTG induce all BL21 (Try 0.5mM iptg, 16C)
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Bacteria culture of Ko Topo 1,2,3, and Tf Topo A,B
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Ligation of SpeI and PstI cut K525998 and sig RFP
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Colony PCR of Ko 1 (11,12), Ko 3(6,7,8,9,10),Tf(1,2,3,4,5)
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August 13th, Tuesday
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Prepare Cm plates
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Run gel of colony PCR products from 12-Aug
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Miniprep of bacteria culture TfB (3,4,5) and KO (2,4,5), digestion test of miniprep products with NdeI and NotI
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Transformation of Sig RFP Topo and 525 into DH10B and Spread Plate
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Harvest All Induced BL21, Run SDS-PAGE
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August 14th, Wednesday
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Bacteria culture of RFP Topo and RFP 525.
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Redo transformation of RFP Topo into DH10B.
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August 15th, Thursday
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Miniprep of bacteria culture from 14/8.
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August 16th, Friday
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Send out 4 plasmids for DNA sequencing.
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Digestion of Ko topo 2,4 Tf and Pet(Commercial vector).
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August 17th, Saturday
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August 18th, Sunday
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August 19th, Monday
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August 20th, Tuesday
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August 21st, Wednesday
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Bacteria culture of Tf pET28a(t).
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Double digestion test of Ko pET28a(t).
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August 22nd, Thursday
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Miniprep of Tf pET28a(t) and digestion test.
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August 23rd, Friday
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Run gel of digestion product from 22/8.
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Spead plates of KO pET28a(t) 2, 4.
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August 24th, Saturday
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August 25th, Sunday
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August 26th, Monday
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Run gel of Digestion Product and purification.
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August 27th, Tuesday
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Preparing BL21 competent cells.
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Bacteria culture.
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August 28th, Wednesday
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Miniprep and digestion test.
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August 29th, Thursday
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Bacteria culture.
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Ligation and transformation into DH10B.
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August 30th, Friday
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Miniprep and Digestion Test.
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August 31st, Saturday
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Bacteria culture.
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September 1st, Sunday
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Miniprep and digestion test.
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Transformation.
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September 2nd, Monday
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September 3rd, Tuesday
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September 4th, Wednesday
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September 5th, Thursday
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September 6th, Friday
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Miniprep Kop ET 28a(t) DH10B and digestion product.
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Digestion and purification of pET28a(t) vector.
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Transformation.
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Bacteria culture.
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September 7th, Saturday
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September 8th, Sunday
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Miniprep and Digestion test.
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September 9th, Monday
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His-tagged protein purification.
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September 10th, Tuesday
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September 11th, Wednesday
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His-tagged protein purification.
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PAGE of the purified protein, coomassie blue staining.
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September 12th, Thursday
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September 13th, Friday
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September 14th, Saturday
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September 15th, Sunday
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September 16th, Monday
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September 17th, Tuesday
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September 18th, Wednesday
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September 19th, Thursday
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September 20th, Friday
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Polyphosphate Assay
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Phosphate Assay
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SigRFP localization of Eut MCP
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Latest revision as of 11:14, 26 September 2013



Shannon

Samson

Ting-Yen

Lai Hei Ming

Charmaine

Fall

Mickey

Kenny

Calvin

Wilson

Samuel