Team:Queens Canada/Notebook/Week16

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Revision as of 22:18, 26 October 2012

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Notebook - Week 16

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Date	Protocol	People	DNA (if relevant)	Quantities and Parameters (if relevant)	Notes on Protocol
12-08-2012	PCR	Beini	GE pFliC1 + GFP + FliC2
12-08-2012	Digestion with XS	Faisal	PCR products SmtA and fMt		Fast Digest, enzymatic purification using Biobasic kit
12-08-2012	PCR overlap extension of MBPs with FliC1	Faisal	SmtA, fMt, FliC1
12-08-2012	Soft agar (0.25%) plates	Beini		made 7 plates each of [C], [amp], [tet]
12-08-2012	Miniprep	Beini	K314100 high exp cassette, K314101 low exp cassette, K314103 lac expression cassette
12-08-2012	Gel	Beini	K081005 SP GE, K314104 SP GE
12-08-2012	Gel	Beini	nFF GFP GE, nFF GFP XP GE, pFF GFP XP GE, MPP K314100, MPP K314101, MPP K314103
12-08-2012	Gel extraction	Kevin	nFF GFP, nFF GFP XP, pFF GFP XP, K081005 SP, K314104 SP
12-08-2012	Gel	Kevin	MPP K081005, MPP K13104, pFF GFP, nFF GFP, pFF GFP XS
12-08-2012	Digestions	Beini	"MPP K314100 with SP
MPP K314103 with SP
GE nFF GFP with XP"	digested MPPs for 12 mins, GEs for 30 mins
12-08-2012	Ligations	Kevin	pFF GFP in 1A2 from K088006	used 6 and 8 uL of vector for 1:1 and 3:1 ligations
12-08-2012	Gel	Kevin	1:1 lig, 3:1 lig, DIG XP nFF GFP, DIG SP K314100, DIG SP K314103		gel extracted the digestions
12-08-2012	Heat Shock Transformation using Motility Plates	Kevin
13-08-2012	Ligations	Victor	"nFF 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"
13-08-2012	Gel	David, Kevin	fMT FliC, SmA FliC, DIG nFF GFP XP, LIG nFF GFP XP 1A3, LIG nFF GFP XP T7, LIG nFF GFP XP K314100
13-08-2012	Digestions	Victor	nFF GFP GE (3) with XP
13-08-2012	Gel extraction	David	fMt FliC, SmtA FliC from above gel
13-08-2012	Gel	Kevin	nFF GFP XP, K314100 SP, SmtA FliC1, fMt FliC1
13-08-2012	Ligations	Kevin	"Vectors: K314104, K081005, K314100, K314003, J04500
Insert: nFF GFP XP"	1:1, 2:1 insert:vector ratios	plated 100 uL on [K], 100 uL on [AK], spun down the rest at 12000 rpm and resuspended 100 uL on [AK]
13-08-2012	Digestions	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
14-08-2012	Liquid cultures	Kevin	pFF GFP 1A3 1:3 lig (the rest)
14-08-2012	Digestions	David	GEs SmtA + FliC, fMt + FliC1 with SpeI
14-08-2012	Digestions	Victor	nFF GFP GE with XP
14-08-2012	Digestions of linearized plasmid backbone	Victor	pSB1C3, pSB1A3 with XP and DpnI		digestion purification
14-08-2012	Gel Extraction	Victor	DIG XP nFF GFP, npp pFF GFP 1A3, DIG EP pFF GFP 1A3, pSB1C3 XP		did enzymatic purification of pSB1C3 digest instead of gel purification
14-08-2012	Gel Extraction	Kevin	nFF GFP XP, nFF GFP XP, DIG xylE + RBS XP
14-08-2012	Miniprep	Kevin	LIG pFF GFP + 1A3 1:3 (the rest)
14-08-2012	Digestion	Kevin	LIG pFF GFP + 1A3 1:3 (the rest) with EP
14-08-2012	PCR overlap extension	Faisal	fliC1 + MBP, fliC2	did 1:1, 2:1, 3:1 ratios of fliC1 + MBP : fliC2	digestion purification of SmtA + fliC1 and fMT + fliC1 digested with SpeI
14-08-2012	Ligations	Victor, Mitangi	pSB1A3 + nFF GFP	did 1:1, 2:1 ratios of insert : vector
14-08-2012	Gel to check for concentrations	Victor	nFF GFP GE XP, nFF GFP GE XP, GE xylE + RBS XP plasmid, GE xylE + RBS
14-08-2012	Liquid cultures of fluorescent colonies	Kevin, Faisal	nFF 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"
14-08-2012	Heat shock transformation	Beini	nFF GFP + 1A3	1:1, 2:1, ctrl
14-08-2012	Gel extraction	Faisal, David	fmt FliC1 + FliC2, SmtA FliC1 + FliC2
15-08-2012	Minipreps	Kevin	nFF GFP + J04500 6 trials, pFF GFP 1A3 1:1 the rest
15-08-2012	PCR of Full FliC MBP Gel Extractions	David, Faisal	fmt FliC1 + FliC2, SmtA FliC1 + FliC2
15-08-2012	Digestion	Phillip	nFF GFP + J04500 6 trials, pFF GFP 1A3 1:1 the rest with ES
15-08-2012	Gel	Victor	"DIG B0015 EX
DIG B0017 EX
DIG nFF GFP + J04500 ES
pFF GFP + 1A3
MPP nFF GFP + J04506
MPP nFF GFP + J04560"
15-08-2012	Ligations	Victor	GE nFF GFP XP, 1C3 XP GE	did 1:1, 2:1 insert:vector ratios
15-08-2012	Gel	Phillip	"MPP nFF + J04500
MPP pFF GFP 1A3"
15-08-2012	Liquid culture	Phillip	nFF GFP + 1A3
15-08-2012	Streak plating	Victor	nFF GFP + J04500	1:2, 1:3, 1:1
15-08-2012	Gel Extraction of FF MBP products from PCR overlap extension	Faisal	SmtA FliC1 + FliC2
15-08-2012	Digestioin	Victor	B0015, B0017 terminators with EX
15-08-2012	PCR overlap extension	Kevin	mCer DP XS, nFF GFP + J04500	"need a super high insert:vector ratio, >>50:1
extension time 5 min 30 s, 17 cycles"
15-08-2012	Liquid Culture	Kevin	nFFGFP 1A3 4 trials, J04500
15-08-2012	Motility plate inoculation	Kevin	nFF GFP + J04500 6 trials	3 uL stab + inject per spot	1 stop only on pipette
15-08-2012	Digestion of mCer OE	Kevin	mCer OE	17 uL DNA, 1 uL DpnI, 2 uL buffer Tango
16-08-2012	Digestion of PP mCer and PP mCit with XS	David	PP mCer, PP mCit		ran out of SpeI, only got a bit of S for mCit1; mCer2 and mCit2 with X only
16-08-2012	Ligations	Victor	GE nFF GFP XP + 1C3 XP GE from Antigen-43	1:1 insert:vector
16-08-2012	Digestion	Victor	Luciferase PCR products with XS
16-08-2012	Miniprep	Kevin	nFF GFP + 1A3, J04500, nFF GFP + 1A3, nFF GFP + 1A3
16-08-2012	Digestion with EP	Kevin	nFF GFP + 1A3, nFF GFP + 1A3, nFF GFP + 1A3	digested 10 uL of dna	NEB
16-08-2012	Gel extraction	Victor, 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
16-08-2012	Gel	Kevin	MPP 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
16-08-2012	Digestion w/ EcoRI-HF	Kevin	MPP nFF GFP 1A3 2:1 the rest
16-08-2012	Gel	Kevin	GE mCer X, GE luciferase XS, DIG nFF GFP E
16-08-2012	PCR overlap extension	Kevin	"inserts: mCer, luciferase
vector: nFF GFP + J04500 K"	8 mins. 30 s extension time, 18 cycles	1 trial with Kapa, 1 trial with Phusion
16-08-2012	Liquid cultures	Kevin	fluorescent colony AK nFF GFP J04500 1:3 the rest from 08/14 streak plate
16-08-2012	Spectrometer	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"
16-08-2012	Motility plate inoculations	Kevin	J04500, nFF GFP + J04500 1:2 K, nFF GFP + J04500 1:2 AK	only J04500 shows motility
16-08-2012	Gel	Phillip	PCR-OE products: luciferase Kapa, luciferase Phusion
17-08-2012	250 mL inoculations of 2XTY	Victor	AK nFF GFP + J04500 1:2, AK J04500
17-08-2012	Digestions with DpnI using Tango buffer	Phillip	PCR-OE products: luciferase Kapa, luciferase Phusion
17-08-2012	Using the ultracentrifuge to obtain a bacterial cell pellet	Victor, as told by Jun			"use JA-10 rotor and 250 mL bottles
10 000 rpm, 6000 speed, 10 mins., 4C"
17-08-2012	Tris-HCl buffer	Victor		"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"
17-08-2012	Liquid culture of ligations	Beini	nFF GFP 1C3 100 pellet, nFF GFP 1C3 100 uL, nFF GFP 1A2 1:1 pellet, nFF GFP 1A2 1:1 100 uL
18-08-2012	Isolation of flagella	Beini	AK 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"
18-08-2012	Liquid cultures	Mitangi	AK DIG luciferase Kapa DpnI
18-08-2012	Miniprep	Mitangi	nFF GFP 1A2, nFF GFP 1C3
18-08-2012	Digestion with PstI	Mitangi	nFF GFP 1A2, nFF GFP 1C3, nFF GFP 1A3 (all MPPs)
18-08-2012	Gel	Beini	MPP 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
18-08-2012	Miniprep	Beini	AK nFF GFP J04500 1:3 the rest		made glycerol stock
18-08-2012	Miniprep	Beini	AK 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.

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-<p>Notebook - Week 1</p>
+<p>Notebook - Week 16</p>
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