Team:Queens Canada/Notebook/Week16



Notebook - Week 16

DateProtocolPeopleDNA (if relevant)Quantities and Parameters (if relevant)Notes on Protocol
08/10/2012Fast DigestionVictorppFGFP + nFFGFP  
08/10/2012Gel ExtractionVictornFFGFP + pFFGFP  
08/10/2012DigestionsKevinJ48200 w/ pSB1AT3 J13601 Lac operator w/ pSB1A3 746007 Antigen 43 w/ pSB1C3  
08/10/2012digestionFaisal and DavidSmtA and FMT with X and S  
08/10/2012Gel ExtractionPhillipIPTG (SP), T7 promotor (SP), mCerFC (1,3,4 - 2 trials each)  
08/10/2012DigestionPhillipGE IPTG SP [X], GE T7 Prom SP [X]  
08/10/2012Gel ElectrophoresisPhillip 1. ladder 2. GE mCerFC 1,2 3. GE mCerFC 1,1 4. GE mCerFC 3,1 5. GE mCerFC 3,2 6. GE mCerFC 4,1 7. GE mCerFC 4,2 8. DIG GE DIG IPTG SP [X] T1 9. DIG GE DIG IPTG SP [X] T2 10. ladder 11. DIG GE DIG T7 Prom SP [X] T1 12. DIG GE DIG T7 Prom SP [X] T1 13. DIG nFFGFP XP 14. DIG nFFGFP 2 XP 15. DIG I15601 (pSB1A3) XP 16. DIG J45200 Banana Odour XP (pSB1AT3) 17. DIG 346002 Antigen 43 XP (pSB1C3) 18. Ladder 
08/10/2012Fast DigestionVictornFFGFP GE 2, ppFGFP GE 1, MPP Bio-timer k088006, MPP I13601, MPP Bis0A K123000, MPP Antigen 43 K346007denatured for 14 mins at 37 degrees. Heat inactivated at 80 degrees for 20 mins 
08/10/2012Gel ExtractionVictor and Kevin 1. ladder 2. nFFGFP DIG XP 3. ppFGFP DIG XP 4. DIG Bio-timer k088006 XP 5. MPP Bis0A K123000 XP T2 6. DIG Antigen 43 K346007 XP 7. DIG KI13601 XP 
08/10/2012PCRPhillipnFFGFP 1+2, ppFGFP 1+225 cycles, 2ul of dna usedhotstart
08/10/2012PCRBeinimCerFC25 cycles, 10uL of dna usedhotstart
08/10/2012GelKevin 1. ladder 7. J04500 SP dig 8. nFFGFP dig XpLanes 2 to 6 are unknown
08/10/2012DigestionPhillipJ04500 SP  
08/10/2012DigestionBeiniGE nFFGFP 1 [XP]  
08/10/2012GelBeini1C3 XP GE, 1A3 BisdA XP GE, I13601 1A2 XP GE, J04500 SP GE, K088006 Timer XP GE pSB1A2, GE pFF GFP XP Small Gel 1
08/10/2012GelBeinimCer FC C, nFF GFP C, T7 prom GE X, IPTG SP GE, DIG GE IPTG SP X, GE T7 prom SP Small Gel 2
08/10//2012Gel extractionBeini, KevinmCer FC, pFF GFP, nFF GFP GE XP, pFF GFP XSloaded 4 uL loading dye : 16 uL DNA; for digests with FD green, loaded all 30 uL 
08/11//2012Liquid culturesKevinK314101 (low exp cassette), K314100 (high conc exp), K314103 (lac ind cassette)3.68 uL [C] added to each 
08/11//2012MiniprepKevinK081005 (cons. prom. + RBS) x 2, K314104 (T7 exp. cassette) x 2  
08/11//2012DigestionsKevinMPPs: K081005, K314104 with SP, GEs: nFF GFP, pFF GFP with XPMPPs: 5 uL DNA, 20 uL total rxn volume, GEs: 15 uL DNA, 30 uL total rxn volume Fast Digest
12/08/2012PCRBeiniGE pFliC1 + GFP + FliC2  
12/08/2012Digestion with XSFaisalPCR products SmtA and fMt Fast Digest, enzymatic purification using Biobasic kit
12/08/2012PCR overlap extension of MBPs with FliC1FaisalSmtA, fMt, FliC1  
12/08/2012Soft agar (0.25%) platesBeini made 7 plates each of [C], [amp], [tet] 
12/08/2012MiniprepBeiniK314100 high exp cassette, K314101 low exp cassette, K314103 lac expression cassette  
12/08/2012GelBeiniK081005 SP GE, K314104 SP GE  
12/08/2012GelBeininFF GFP GE, nFF GFP XP GE, pFF GFP XP GE, MPP K314100, MPP K314101, MPP K314103  
12/08/2012Gel extractionKevinnFF GFP, nFF GFP XP, pFF GFP XP, K081005 SP, K314104 SP  
12/08/2012GelKevinMPP K081005, MPP K13104, pFF GFP, nFF GFP, pFF GFP XS  
12/08/2012DigestionsBeiniMPP K314100 with SP, MPP K314103 with SP, GE nFF GFP with XPdigested MPPs for 12 mins, GEs for 30 mins 
12/08/2012LigationsKevinpFF GFP in 1A2 from K088006used 6 and 8 uL of vector for 1:1 and 3:1 ligations 
12/08/2012GelKevin1:1 lig, 3:1 lig, DIG XP nFF GFP, DIG SP K314100, DIG SP K314103 gel extracted the digestions
12/08/2012Heat Shock Transformation using Motility PlatesKevin   
13/08/2012LigationsVictornFF GFP XP GE + 1A3 XP GE. nFF GFP XP GE + 1A3 XP GE, nFF GFP XP GE + T7 GE SP, nFF GFP XP GE + K314100 GE SP, nFF GFP ctrl  
13/08/2012GelDavid, KevinfMT FliC, SmA FliC, DIG nFF GFP XP, LIG nFF GFP XP 1A3, LIG nFF GFP XP T7, LIG nFF GFP XP K314100  
13/08/2012DigestionsVictornFF GFP GE (3) with XP  
13/08/2012Gel extractionDavidfMt FliC, SmtA FliC from above gel  
13/08/2012GelKevinnFF GFP XP, K314100 SP, SmtA FliC1, fMt FliC1  
13/08/2012LigationsKevinVectors: K314104, K081005, K314100, K314003, J04500, Insert: nFF GFP XP1:1, 2:1 insert:vector ratiosplated 100 uL on [K], 100 uL on [AK], spun down the rest at 12000 rpm and resuspended 100 uL on [AK]
13/08/2012DigestionsBeiniMPP J33204 xylE + RBS with XP, nFF GFP GE with XPdigested MPP for 13 mins at 37C, digested GEs for 30 mins at 37Ciced while waiting for 80C bath to heat up
14/08/2012Liquid culturesKevinpFF GFP 1A3 1:3 lig (the rest)  
14/08/2012DigestionsDavidGEs SmtA + FliC, fMt + FliC1 with SpeI  
14/08/2012DigestionsVictornFF GFP GE with XP  
14/08/2012Digestions of linearized plasmid backboneVictorpSB1C3, pSB1A3 with XP and DpnI digestion purification
14/08/2012Gel ExtractionVictorDIG XP nFF GFP, npp pFF GFP 1A3, DIG EP pFF GFP 1A3, pSB1C3 XP did enzymatic purification of pSB1C3 digest instead of gel purification
14/08/2012Gel ExtractionKevinnFF GFP XP, nFF GFP XP, DIG xylE + RBS XP  
14/08/2012MiniprepKevinLIG pFF GFP + 1A3 1:3 (the rest)  
14/08/2012DigestionKevinLIG pFF GFP + 1A3 1:3 (the rest) with EP  
14/08/2012PCR overlap extensionFaisalfliC1 + MBP, fliC2did 1:1, 2:1, 3:1 ratios of fliC1 + MBP : fliC2digestion purification of SmtA + fliC1 and fMT + fliC1 digested with SpeI
14/08/2012LigationsVictor, MitangipSB1A3 + nFF GFPdid 1:1, 2:1 ratios of insert : vector 
14/08/2012Gel to check for concentrationsVictornFF GFP GE XP, nFF GFP GE XP, GE xylE + RBS XP plasmid, GE xylE + RBS  
14/08/2012Liquid cultures of fluorescent coloniesKevin, FaisalnFF GFP + J04500 (6 trials + 3 ctrls)# of fluorescent colonies , AK 1:1 100 uL - 10,AK 1:1 the rest - 21, K 1:1 100 uL - 50, AK: 1:3 100 uL - 0, AK 1:3 the rest - 2, K 1:3 100 uL - 2 
14/08/2012Heat shock transformationBeininFF GFP + 1A31:1, 2:1, ctrl 
14/08/2012Gel extractionFaisal, Davidfmt FliC1 + FliC2, SmtA FliC1 + FliC2  
15/08/2012MiniprepsKevinnFF GFP + J04500 6 trials, pFF GFP 1A3 1:1 the rest  
15/08/2012PCR of Full FliC MBP Gel ExtractionsDavid, Faisalfmt FliC1 + FliC2, SmtA FliC1 + FliC2  
15/08/2012DigestionPhillipnFF GFP + J04500 6 trials, pFF GFP 1A3 1:1 the rest with ES  
15/08/2012GelVictorDIG B0015 EX, DIG B0017 EX. DIG nFF GFP + J04500 ES, pFF GFP + 1A3, MPP nFF GFP + J04506, MPP nFF GFP + J04560  
15/08/2012LigationsVictorGE nFF GFP XP, 1C3 XP GEdid 1:1, 2:1 insert:vector ratios 
15/08/2012GelPhillipMPP nFF + J04500, MPP pFF GFP 1A3  
15/08/2012Liquid culturePhillipnFF GFP + 1A3  
15/08/2012Streak platingVictornFF GFP + J045001:2, 1:3, 1:1 
15/08/2012Gel Extraction of FF MBP products from PCR overlap extensionFaisalSmtA FliC1 + FliC2  
15/08/2012DigestioinVictorB0015, B0017 terminators with EX  
15/08/2012PCR overlap extensionKevinmCer DP XS, nFF GFP + J04500need a super high insert:vector ratio, >>50:1 extension time 5 min 30 s, 17 cycles 
15/08/2012Liquid CultureKevinnFFGFP 1A3 4 trials, J04500  
15/08/2012Motility plate inoculationKevinnFF GFP + J04500 6 trials3 uL stab + inject per spot1 stop only on pipette
15/08/2012Digestion of mCer OEKevinmCer OE17 uL DNA, 1 uL DpnI, 2 uL buffer Tango 
16/08/2012Digestion of PP mCer and PP mCit with XSDavidPP mCer, PP mCit ran out of SpeI, only got a bit of S for mCit1; mCer2 and mCit2 with X only
16/08/2012LigationsVictorGE nFF GFP XP + 1C3 XP GE from Antigen-431:1 insert:vector 
16/08/2012DigestionVictorLuciferase PCR products with XS  
16/08/2012MiniprepKevinnFF GFP + 1A3, J04500, nFF GFP + 1A3, nFF GFP + 1A3  
16/08/2012Digestion with EPKevinnFF GFP + 1A3, nFF GFP + 1A3, nFF GFP + 1A3digested 10 uL of dnaNEB
16/08/2012Gel extractionVictor, BeiniDIG mCer XS, DIG mCer X, dig mCit XS, DIG mCit X, DIG luciferase XS, gelled to check concs: GE nFF GFP XP, GE xylE + RBS XP plasmid, GE xylE + RBS XP DIG mCit XS did not show up on gel
16/08/2012GelKevinMPP nff GFP 2:1 the rest, DIG EP nFF GFP 2:1 the rest, MPP J04500, MPP nFF GFP 1A3, DIG EP nFF GFP 1A3  
16/08/2012Digestion w/ EcoRI-HFKevinMPP nFF GFP 1A3 2:1 the rest  
16/08/2012GelKevinGE mCer X, GE luciferase XS, DIG nFF GFP E  
16/08/2012PCR overlap extensionKevininserts: mCer, luciferase, vector: nFF GFP + J04500 K8 mins. 30 s extension time, 18 cycles1 trial with Kapa, 1 trial with Phusion
16/08/2012Liquid culturesKevinfluorescent colony AK nFF GFP J04500 1:3 the rest from 08/14 streak plate  
16/08/2012SpectrometerKevin OD600, 2XTY against no reference: 0.055, J04500 culture: 1.878, nFF GFP + J04500 1:2 K: 1.701 (using a different cuvette = 1.731),nFF GFP + J04500 1:2 AK: 1.760 
16/08/2012Motility plate inoculationsKevinJ04500, nFF GFP + J04500 1:2 K, nFF GFP + J04500 1:2 AKonly J04500 shows motility 
16/08/2012GelPhillipPCR-OE products: luciferase Kapa, luciferase Phusion  
17/08/2012250 mL inoculations of 2XTYVictorAK nFF GFP + J04500 1:2, AK J04500  
17/08/2012Digestions with DpnI using Tango bufferPhillipPCR-OE products: luciferase Kapa, luciferase Phusion  
17/08/2012Using the ultracentrifuge to obtain a bacterial cell pelletVictor, as told by Jun  use JA-10 rotor and 250 mL bottles 10 000 rpm, 6000 speed, 10 mins., 4C
17/08/2012Tris-HCl bufferVictor 12.1 g Tris, 800 mL H2O, 7.7 mL HCl until pH 7.8 is reached, fill the rest to 1 L of solution, autoclave at Liquid 1 setting 
17/08/2012Liquid culture of ligationsBeininFF GFP 1C3 100 pellet, nFF GFP 1C3 100 uL, nFF GFP 1A2 1:1 pellet, nFF GFP 1A2 1:1 100 uL  
18/08/2012Isolation of flagellaBeiniAK nFF GFP + J04500 1:2, AK J04500 resuspended cell pellet in 100 mL Tris-HCl, blended 1 min 30 s in Waring blender at 4C, centrifuged 10 mins. at 4C, 10 000 rpm, speed 6000 , centrifuged supernatant for 1 h at 4C, 20 000 rpm, speed 6000
18/08/2012Liquid culturesMitangiAK DIG luciferase Kapa DpnI  
18/08/2012MiniprepMitanginFF GFP 1A2, nFF GFP 1C3  
18/08/2012Digestion with PstIMitanginFF GFP 1A2, nFF GFP 1C3, nFF GFP 1A3 (all MPPs)  
18/08/2012GelBeiniMPP nFF GFP 1A3, DIG P nFF GFP 1A3, MPP nFF GFP 1A2, DIG nFF GFP 1A2 P, MPP nFF GFP 1A2, DIG P nFF GFP 1C3, DIG P nFF GFO 1C3  
18/08/2012MiniprepBeiniAK nFF GFP J04500 1:3 the rest made glycerol stock
18/08/2012MiniprepBeiniAK DIG luciferase Kapa DpnI (PCR OE of luciferase + nFF GFP J04500) 

Given that the vast majority of our work now is in the lab, we will only be updating the "Labwork" section of the Notebook.