|
|
| Line 5: |
Line 5: |
| | <html> | | <html> |
| | <body> | | <body> |
| - | <style type="text/css"> | + | <div id="content1"> |
| - | #contentpicture{
| + | <font face="arial size="5"> |
| - | background-color:transparent;
| + | Shannon<br><br> |
| - | width:280px;
| + | Samson<br><br> |
| - | margin:10 auto;
| + | Ting-Yen<br><br> |
| - | float:left;
| + | Lai Hei Ming<br><br> |
| | + | Charmine<br><br> |
| | + | Fall<br><br> |
| | + | Mickey<br><br> |
| | + | Kenny<br><br> |
| | + | Calvin<br><br> |
| | + | Samuel<br><br> |
| | | | |
| - | }
| |
| - | #contenttext{
| |
| - | background-color:transparent;
| |
| - | width:500px;
| |
| - | margin:10 auto;
| |
| - | float:left;
| |
| - | padding:10px;
| |
| - |
| |
| - | }
| |
| - | #contentgap{
| |
| - | background-color:transparent;
| |
| - | width:20px;
| |
| - | margin:0 auto;
| |
| - | float:left;
| |
| - | }
| |
| - | </style>
| |
| - | <div id="wrapper">
| |
| - | <div id="content1">
| |
| - |
| |
| - |
| |
| - | <p><font face="Arial" size="5">Talk delivered to Sec School Students</font></p><br>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - |
| |
| - | <br><br>
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | <p>
| |
| - | <font face="Arial" size="2">
| |
| - | June 28th, Friday
| |
| - | Long Amp PCR + Cloning
| |
| - | Cargo Team: (Only KO genome available)
| |
| - | Native Ko_ppk Amplified > Gel Purified (49ng/ul) > TA vector Ligation+Transformation > Spread Plate
| |
| - | 1st step Signal Ko_ppk Am
| |
| - | Surface Team:
| |
| - | plified > Gel Purified (weak) > 2nd PCR
| |
| - | Native EutS Amplified > Gel Purified (25ug/ul) > TA vector Ligation+Transformation > Spread Plate
| |
| - | His_EutS Amplified > Gel Purified > 2nd PCR
| |
| - | Flag_EutS Amplified > Gel Purifed > 2nd PCR
| |
| - | Ko native ppk and Ko sigppk 1st PCR product.
| |
| - |
| |
| - | June 29th, Saturday
| |
| - | Long Amp PCR + Cloning, continuous PCR and TA vector transformation.
| |
| - |
| |
| - | June 30th, Sunday
| |
| - | His Euts TA colonies for culture.
| |
| - | Redo transformation of native EutS and Flag EutS.
| |
| - | Continuous PCR of Tf_ppk1 and Ko_ppk1.
| |
| - |
| |
| - | July 1st, Monday
| |
| - | Miniprep of HisEutS and Ko_ppk1 TA clone culture and Digestion test.
| |
| - | Overnight ligation of Native Ko_ppk1, Native Tf_ppk1, pLacHisEutS, Sig_Koppk1(Weak), Flag_EutS
| |
| - |
| |
| - | July 2nd, Tuesday
| |
| - | Mass Digestion of His_EutS.
| |
| - | Continuous PCR and gel purification.
| |
| - | Miniprep for EutS Topo conlonies.
| |
| - | 4 Transfromation (Topo Clone) : Native Ko_ppk1, Native Tf_ppk1, pLacHisEutS, Sig_Koppk1(Weak), Flag_EutS
| |
| - |
| |
| - | July 3rd, Wednesday
| |
| - | Continuous PCR of Ko_sigppk, His_EutSMNLK and Flag_EutSMNLK.
| |
| - | Mass Digestion of native EutS.
| |
| - | Transformation: His_Eut into K525998 (Cm Plate); TF_sigppk into Topo (Amp+Xgal Plate); Flag_EutS into Topo (1),(2)
| |
| - |
| |
| - | July 4th, Thursday
| |
| - | MiniPrep and Digestion Test: Native Ko_ppk1, Native Tf_ppk1, Sig_Koppk1
| |
| - | Transformation: NativeEutS into 525 (1,2) Cm Plate; Native Tfppk, Koppk, sig_koppk
| |
| - | Prepare DH10B Competent Cells.
| |
| - |
| |
| - | July 5th, Friday
| |
| - | Miniprep and Digestion Test: HisEutS 525, FlagEutS topo, Tf_sigppk topo.
| |
| - | 2nd Round PCR for His,Flag EutSMNLK
| |
| - |
| |
| - | July 6th, Saturday; July 7th, Sunday; July 8th, Monday
| |
| - | A very Tough Weekend (6-8/7)
| |
| - | PCR Native Ko, Native Tf, Sig Tf and Sig Ko was ligated in to Topo Vector.
| |
| - | Many White Colonies were observed. (Also with a few Blue Colonies)
| |
| - | Very HAPPY about the result and picked colonies for culture.
| |
| - | Culture Two Times and Found out the growth rate was abnormally Slow and the Culture was not cloudy and contain fillament like clusters.
| |
| - | Maybe the ppk gene might be toxic to cell?<< Wrong
| |
| - | Maybe the shaker speed and temperature not correct? << Wrong
| |
| - | WE have Fungus/other bacteria Comtamination!!!
| |
| - | Hints:
| |
| - | Colonies morphology>>very white and not transparent and moist compared to Ecoli colonies
| |
| - | Culture grow well (withou filament clusture) in 32C.
| |
| - | Can grow in Wrong Antibiotics. (Topo vector is Kana resistant, I use Cm to test>Still grow)
| |
| - | Tried to miniprep on tube with less clusters. No Plasmid.
| |
| - | Right Now We don't Know the source of contamination.
| |
| - | Overview:
| |
| - | What we have:
| |
| - | We have EutS, His EutS, Flag EutS in Topo Vector. Positive Digestion Pattern.
| |
| - | Send for sequencing on 9/7.
| |
| - | To avoid comtaminantion.
| |
| - | Will Redo Transformation to create a new stock.
| |
| - | But Right now, we have:
| |
| - | HisEutS 1,2 glycerol stock and streak plates.
| |
| - | FlagEutS 1,2 glycerol stock and streak plates.
| |
| - | (picked colonies from plates and tested postive, but cannot tell whether there is comtamination)
| |
| - | EutS Retransformed plates and glycerol stock.
| |
| - |
| |
| - | Previous EutS, His_EutS, Flag_EutS in to 525 vector failed. Will try again.
| |
| - |
| |
| - | But we still don't have any ppk in vector!! But have succeed in PCR (But result random)
| |
| - |
| |
| - | Cannot Amplify EutSMNLK from K311004. Will send K311004 for sequencing.
| |
| - |
| |
| - |
| |
| - | July 9th, Tuesday
| |
| - | Miniprep and Digestion Test of Tf native ppk, Tf sigppk and Ko sigppk.
| |
| - | Redo ligation and transformation of Ko native ppk, Ko sigppk and Tf sigppk.
| |
| - |
| |
| - | July 10th, Wednesday
| |
| - | PCR of Ko native ppk, Tf sigppk, Ko sigppk
| |
| - | Tranformation of 4 kind of ppk1.
| |
| - | Mass Digestion of Tf native ppk plasmid 1,2
| |
| - | Overnight culture of Tf native ppk1.
| |
| - |
| |
| - | July 11th, Thursday
| |
| - | Colony PCR.
| |
| - | MiniPrep of three Tf native ppk
| |
| - | Making competent cells.
| |
| - | Ligation of Tf native DNA (XbaI/ PstI) with 525 vector.
| |
| - | Transformation of Tf sigppk.
| |
| - |
| |
| - | July 12th, Friday
| |
| - | 1st round PCR of KO sigppk1
| |
| - | Miniprep of 12 tubes; Digestion test
| |
| - |
| |
| - | July 13th, Saturday
| |
| - | Digestion test.
| |
| - | Picked EutS 4, HisEutS 1, FlagEutS 1, Tfppk 2 plasmid transformed into BL21
| |
| - | Colony PCR of sigppk.
| |
| - |
| |
| - | July 14th, Sunday
| |
| - | 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.
| |
| - | Digestion of Tf sigppk plasmid (XbaI and PstI) and purify 2100bp tf sigpkk DNA → Overnight ligation with 525 vector.
| |
| - | Ligation of Ko sigppk PCR product into topo vector → Trasformation
| |
| - |
| |
| - | July 15th, Monday
| |
| - | Miniprep and Digestion Test: Ko native ppk.
| |
| - | Transformation of Tf sigppk 525 vector (into Cm plate), Ko sigppk topo vector (into Kan plate)
| |
| - | Pick two colonies from Tf sigppk stock plate and culture overnight.
| |
| - |
| |
| - | July 16th, Tuesday
| |
| - | Check plates (Tf sigppk 525, Ko sigppk topo) and colonies PCR, culture 6 colonies.
| |
| - | Digestion Ko native ppk overnight.
| |
| - |
| |
| - | July 17th, Wednesday
| |
| - | Miniprep and digestion test of Topo Ko sig 1,2,3 and Tf sigppk 525 1,2,3.
| |
| - | Digestion overnight (Ko sigppk 3, Tf sigppk1 and K525998 plasmids)
| |
| - | Gel Purification of digestion product.
| |
| - |
| |
| - | July 18th, Thursday
| |
| - | Run gel and purification of Digestion Product.
| |
| - | Ligation of Ko native ppk and Tf sigppk to new 525 vector, transformation.
| |
| - | Transformation of Ko native ppk/525 into DH10B, spread plate.
| |
| - | Pick two colonies from Ko native ppk and one colony from Ko sigppk stock plates and culture overnight.
| |
| - |
| |
| - | July 19th, Friday
| |
| - | Colonies PCR of Ko native ppk 525, Tf sigppk 525.
| |
| - | MiniPrep of yesterday's culture. ( Ko native ppk topo and Ko sigppk topo)
| |
| - | Pick 2 colonies from Euts native525, 2 from Flag Euts525, 2 from His Euts525, 1 from Tf native ppk525 and culture overnight.
| |
| - |
| |
| - | July 20th, Saturday
| |
| - | Antibiotics Mistake, all culture failed. Redo culture again.
| |
| - |
| |
| - | July 21st, Sunday
| |
| - | MiniPrep and digestion test of Ko native ppk 525, Tf sigppk525.
| |
| - | Transformation of Ko native ppk 525 vector into Bl21 and spread plates.
| |
| - | Culture Tf native ppk 525 BL21 overnight. Culture 4 tubes of K311004 DH10B overnight.
| |
| - |
| |
| - | July 22nd, Monday
| |
| - | Check plates: No colonies. Thus redo transformation of Ko native ppk 525 vector into Bl21.
| |
| - | Miniprep of K311004 DH10B, concentrate into one plasmid purification kit. Get a high conc. plasmid sample (110.4ng/ul)
| |
| - | IPTF induction of Tf native ppk Bl21, culture overnight.
| |
| - |
| |
| - | July 23rd, Tuesday
| |
| - | Run PAGE of induced Tf native ppk Bl21 cell lysate and control, coomassie blue staining.
| |
| - |
| |
| - | July 24th, Wednesday
| |
| - | As yesterday's result was not ideal, redo PAGE and coomassie blue staining.
| |
| - |
| |
| - | July 25th, Thursday
| |
| - | Check the coomassie blue staining result: A 500kDa band (referring to the sequencing result, it might be a premature stop codon. normal ppk: 78kDa)
| |
| - | 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)
| |
| - | PCR test: plasmid Sigppk1 topo 1, Plasmid sigppk1 topo 2
| |
| - | Culture Ko sigppk topo 1,2,3 overnight.
| |
| - | Culture Tf native ppk bl21 ovenight.
| |
| - |
| |
| - | July 26th, Friday
| |
| - | IPTG inductionof Tf native ppk bl21(10, 20).
| |
| - | Miniprep and Digestion test of Ko sigppk topo 1,2,3.
| |
| - | Run gel of Ko sigppk 3 PCR product.
| |
| - | Ligation of Tf sigppk and Ko native ppk into topo vector and transformation
| |
| - | Culture Euts native, Flag, His Bl21 overnight.
| |
| - |
| |
| - | July 27th, Saturday
| |
| - | Run PAGE of Tf native ppk bl21, coomassie blue staining.
| |
| - | IPTG induction of Euts Native, Flag, His.
| |
| - | Check plates. (White colonies, Contamination?)
| |
| - |
| |
| - | July 28th, Sunday
| |
| - | Colony PCR of TF sigppk and Ko native ppk topo DH10B
| |
| - | Run PAGE of Euts Native, Flag, His Bl21. Coomassie Blue staining.
| |
| - | Transformation of Tf sigppk topo and spread plate.
| |
| - |
| |
| - | July 29th, Monday
| |
| - | Check plate of Tf sigppk topo, colony PCR.
| |
| - | Miniprep of Ko native ppk topo and Digestion test.
| |
| - | IPTG induction of Tf native ppk.
| |
| - | Transformation of Euts native, flag, his; Tf sigppk 1, 5 (all in 525 vector) into BL21 and spread plates.
| |
| - |
| |
| - | July 30th, Tuesday
| |
| - | Run gel of digestion product, purification. Ligation into 525 vector.
| |
| - | Run PAGE of Tf native ppk and coomassie blue staining.
| |
| - | Miniprep of Tf sigppk topo.
| |
| - | Culture Euts native, flag, his; Tf sigppk 1, 5 BL21 colonies overnight for IPTG induction.
| |
| - |
| |
| - | July 31st, Wednesday
| |
| - | IPTG induction of Euts native, flag, his; Tf sigppk 1, 5
| |
| - | Digestion of Tf sigppk topo and digestion test. Ligation into 525 vector.
| |
| - | Transformation of Ko native ppk 525 into DH10B and spread plates.
| |
| - |
| |
| - |
| |
| - | August 1st, Thursday
| |
| - | Run PAGE gel of Euts native, flag, his; Tf sigppk 1, 5 and Coomassie blue staining.
| |
| - | Transformation of Tf sigppk 525 into DH10B and spread plate.
| |
| - | Colony PCR of Ko native ppk 525 DH10B
| |
| - | 1st Round PCR of Tf sigppk.
| |
| - |
| |
| - | August 2nd, Friday
| |
| - | Miniprep and digestion test.
| |
| - | Colony PCR.
| |
| - |
| |
| - | August 3rd, Saturday
| |
| - | Miniprep of Tf sigppk, Ko native ppk in DH10B ,tf native ppk 5, native EutS, Flag EutS, His EutS in BL21. Digestion test.
| |
| - | Try IPTG induction again using a new condition.
| |
| - | Transformation of Tf sigppk 2,5, 7 in BL21.
| |
| - |
| |
| - | August 4th, Sunday
| |
| - | PAGE of EutS native, Flag EutS and HisEutS.
| |
| - |
| |
| - | August 5th, Monday
| |
| - | August 6th, Tuesday
| |
| - | August 7th, Wednesday
| |
| - | August 8th, Thursday
| |
| - | August 9th, Friday
| |
| - | August 10th, Saturday
| |
| - | PCR ppk for commercial vector(adding tag for purification)
| |
| - | PCR sig-RFP for detecting localization
| |
| - | Transformation of KO525 (4, 19) into bl21
| |
| - | Transformation of ligation product (RFP into 525)
| |
| - | 1st PCR of Sig Ko
| |
| - |
| |
| - | August 11th, Sunday
| |
| - | Colony PCR of RFP-525 plates, culture positive colonies
| |
| - | Gel Purify 1st PCR SigKo, 2nd PCR
| |
| - | Transform Ko525(4), Ko525(19) into DH5a for DNA storage
| |
| - | Overnight Culture BL21: KO525(4)(19),TFsig(2),TF(6)(7)(8)
| |
| - |
| |
| - | Transform Ko Topo 1, Ko Topo 2, Ko Topo 3, Tf Topo A, Tf topo B into BL21 and spread plates and grow overnight.
| |
| - | Second PCR sig-RFP, Purified and digest using S+P overnight
| |
| - | Digest 525948 Vector using S+P overnight
| |
| - | Ligatation of sig 2nd PCR product and TOPO
| |
| - |
| |
| - | August 12th, Monday
| |
| - | Miniprep RFP525 (2,4,5,6)>>Digestion test>>Transform (4) into BL21
| |
| - | Gel purify 2nd SigKo, SigRFP >> Ligate into T-vector>> Transform DH5a
| |
| - | Transform Pet28a (kana R) into DH5a to get vector
| |
| - | Transform K311004,HisSMNLK,FlagSMNLK into BL21
| |
| - | Streak HisSMNLK (1a),FlagSMNLK(4b) DH5a to get DNA
| |
| - | Culture NativeEutS, HisS, FlagS 525 to get DNA
| |
| - | IPTG induce all BL21 (Try 0.5mM iptg, 16C)
| |
| - | Bacteria culture of Ko Topo 1,2,3, and Tf Topo A,B
| |
| - | Ligation of SpeI and PstI cut K525998 and sig RFP
| |
| - | Colony PCR of Ko 1 (11,12), Ko 3(6,7,8,9,10),Tf(1,2,3,4,5)
| |
| - |
| |
| - | August 13th, Tuesday
| |
| - | Prepare Cm plates
| |
| - | Run gel of colony PCR products from 12-Aug
| |
| - | Miniprep of bacteria culture TfB (3,4,5) and KO (2,4,5), digestion test of miniprep products with NdeI and NotI
| |
| - | Transformation of Sig RFP Topo and 525 into DH10B and Spread Plate
| |
| - | Harvest All Induced BL21, Run SDS-PAGE
| |
| - |
| |
| - | August 14th, Wednesday
| |
| - | Bacteria culture of RFP Topo and RFP 525.
| |
| - | Redo transformation of RFP Topo into DH10B.
| |
| - |
| |
| - | August 15th, Thursday
| |
| - | Miniprep of bacteria culture from 14/8.
| |
| - |
| |
| - | August 16th, Friday
| |
| - | Send out 4 plasmids for DNA sequencing.
| |
| - | Digestion of Ko topo 2,4 Tf and Pet(Commercial vector).
| |
| - |
| |
| - | August 17th, Saturday
| |
| - | August 18th, Sunday
| |
| - | August 19th, Monday
| |
| - | August 20th, Tuesday
| |
| - | August 21st, Wednesday
| |
| - | Bacteria culture of Tf pET28a(t).
| |
| - | Double digestion test of Ko pET28a(t).
| |
| - |
| |
| - | August 22nd, Thursday
| |
| - | Miniprep of Tf pET28a(t) and digestion test.
| |
| - |
| |
| - | August 23rd, Friday
| |
| - | Run gel of digestion product from 22/8.
| |
| - | Spead plates of KO pET28a(t) 2, 4.
| |
| - | August 24th, Saturday
| |
| - | August 25th, Sunday
| |
| - | August 26th, Monday
| |
| - | Run gel of Digestion Product and purification.
| |
| - |
| |
| - | August 27th, Tuesday
| |
| - | Preparing BL21 competent cells.
| |
| - | Bacteria culture.
| |
| - |
| |
| - | August 28th, Wednesday
| |
| - | Miniprep and digestion test.
| |
| - |
| |
| - | August 29th, Thursday
| |
| - | Bacteria culture.
| |
| - | Ligation and transformation into DH10B.
| |
| - |
| |
| - | August 30th, Friday
| |
| - | Miniprep and Digestion Test.
| |
| - |
| |
| - | August 31st, Saturday
| |
| - | Bacteria culture.
| |
| - |
| |
| - | September 1st, Sunday
| |
| - | Miniprep and digestion test.
| |
| - | Transformation.
| |
| - |
| |
| - | September 2nd, Monday
| |
| - | September 3rd, Tuesday
| |
| - | September 4th, Wednesday
| |
| - | September 5th, Thursday
| |
| - | September 6th, Friday
| |
| - | Miniprep Kop ET 28a(t) DH10B and digestion product.
| |
| - | Digestion and purification of pET28a(t) vector.
| |
| - | Transformation.
| |
| - | Bacteria culture.
| |
| - |
| |
| - | September 7th, Saturday
| |
| - | September 8th, Sunday
| |
| - | Miniprep and Digestion test.
| |
| - |
| |
| - | September 9th, Monday
| |
| - | His-tagged protein purification.
| |
| - |
| |
| - | September 10th, Tuesday
| |
| - |
| |
| - | September 11th, Wednesday
| |
| - | His-tagged protein purification.
| |
| - | PAGE of the purified protein, coomassie blue staining.
| |
| - |
| |
| - | September 12th, Thursday
| |
| - | September 13th, Friday
| |
| - | September 14th, Saturday
| |
| - | September 15th, Sunday
| |
| - | September 16th, Monday
| |
| - | September 17th, Tuesday
| |
| - | September 18th, Wednesday
| |
| - | September 19th, Thursday
| |
| - | September 20th, Friday
| |
| - | Polyphosphate Assay
| |
| - | Phosphate Assay
| |
| - | SigRFP localization of Eut MCP
| |
| - |
| |
| - | </font>
| |
| - | </p>
| |
| - |
| |
| - | <br><br>
| |
| - |
| |
| - |
| |
| - |
| |
| - | </div>
| |
| - | </div>
| |
| | | | |
| | </body> | | </body> |
| | </html> | | </html> |
| | {{:Team:Hong_Kong_HKU/templates/footer}} | | {{:Team:Hong_Kong_HKU/templates/footer}} |
"
