Team:Queens Canada/Notebook/Week16


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Notebook - Week 1

DateProtocolPeopleDNA (if relevant)Quantities and Parameters (if relevant)Notes on Protocol
12-08-2012PCRBeiniGE pFliC1 + GFP + FliC2  
12-08-2012Digestion with XSFaisal PCR 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%) plates Beini 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  
12-08-2012Digestions Beini"MPP K314100 with SP
MPP K314103 with SP
GE nFF GFP with XP"digested 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 
12-08-2012Gel Kevin1:1 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   
13-08-2012LigationsVictor"nFF GFP XP GE + 1A3 XP GE
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  
13-08-2012Digestions VictornFF 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  
13-08-2012LigationsKevin"Vectors: K314104, K081005, K314100, K314003, J04500
Insert: nFF GFP XP"1:1, 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]
13-08-2012Digestions Beini"MPP J33204 xylE + RBS with XP
nFF GFP GE with XP""digested MPP for 13 mins at 37C
digested GEs for 30 mins at 37C"iced while waiting for 80C bath to heat up
14-08-2012Liquid cultures KevinpFF GFP 1A3 1:3 lig (the rest)   
14-08-2012Digestions David GEs SmtA + FliC, fMt + FliC1 with SpeI  
14-08-2012Digestions VictornFF 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 + 1A3 1:1, 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  
15-08-2012GelVictor"DIG 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  
15-08-2012GelPhillip"MPP nFF + J04500
MPP pFF GFP 1A3"  
15-08-2012Liquid culturePhillipnFF GFP + 1A3   
15-08-2012Streak platingVictornFF GFP + J04500 1:2, 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 + J04500"need 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 inoculation KevinnFF GFP + J04500 6 trials3 uL stab + inject per spot1 stop only on pipette
15-08-2012Digestion of mCer OE KevinmCer 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, Beini"DIG 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 extensionKevin"inserts: mCer, luciferase
vector: nFF GFP + J04500 K"8 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  
16-08-2012Spectrometer Kevin "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 AK only J04500 shows motility 
16-08-2012GelPhillipPCR-OE products: luciferase Kapa, luciferase Phusion  
17-08-2012250 mL inoculations of 2XTYVictor AK 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.