Team:Queens Canada/Notebook/Week16

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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
08/10/2012	Fast Digestion	Victor	ppFGFP + nFFGFP
08/10/2012	Gel Extraction	Victor	nFFGFP + pFFGFP
08/10/2012	Digestions	Kevin	J48200 w/ pSB1AT3 J13601 Lac operator w/ pSB1A3 746007 Antigen 43 w/ pSB1C3
08/10/2012	digestion	Faisal and David	SmtA and FMT with X and S
08/10/2012	Gel Extraction	Phillip	IPTG (SP), T7 promotor (SP), mCerFC (1,3,4 - 2 trials each)
08/10/2012	Digestion	Phillip	GE IPTG SP [X], GE T7 Prom SP [X]
08/10/2012	Gel Electrophoresis	Phillip		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
08/10/2012	Fast Digestion	Victor	nFFGFP GE 2, ppFGFP GE 1, MPP Bio-timer k088006, MPP I13601, MPP Bis0A K123000, MPP Antigen 43 K346007	denatured for 14 mins at 37 degrees. Heat inactivated at 80 degrees for 20 mins
08/10/2012	Gel Extraction	Victor 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
08/10/2012	PCR	Phillip	nFFGFP 1+2, ppFGFP 1+2	25 cycles, 2ul of dna used	hotstart
08/10/2012	PCR	Beini	mCerFC	25 cycles, 10uL of dna used	hotstart
08/10/2012	Gel	Kevin		1. ladder 7. J04500 SP dig 8. nFFGFP dig Xp	Lanes 2 to 6 are unknown
08/10/2012	Digestion	Phillip	J04500 SP
08/10/2012	Digestion	Beini	GE nFFGFP 1 [XP]
08/10/2012	Gel	Beini	1C3 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
08/10/2012	Gel	Beini	mCer 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//2012	Gel extraction	Beini, Kevin	mCer FC, pFF GFP, nFF GFP GE XP, pFF GFP XS	loaded 4 uL loading dye : 16 uL DNA; for digests with FD green, loaded all 30 uL
08/11//2012	Liquid cultures	Kevin	K314101 (low exp cassette), K314100 (high conc exp), K314103 (lac ind cassette)	3.68 uL [C] added to each
08/11//2012	Miniprep	Kevin	K081005 (cons. prom. + RBS) x 2, K314104 (T7 exp. cassette) x 2
08/11//2012	Digestions	Kevin	MPPs: K081005, K314104 with SP, GEs: nFF GFP, pFF GFP with XP	MPPs: 5 uL DNA, 20 uL total rxn volume, GEs: 15 uL DNA, 30 uL total rxn volume	Fast Digest
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.