Team:Aalto-Helsinki/LabBook

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• Week 1

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Lab Book

Before reading our lab notes, familiarize yourelf with our excel sheet, which specifies the nomenclature we've used in out project. You can also find all of our primers on this excel.

Week 1

Worked With	WorkFlow						May 18th - May 22nd
Preparations Plates	Making Cm plates	Stored in +4°C
Competent Cells	Preparing solutions for competent cells	Stored in +4°C
	o/n culture of E. coli TOP10	Chemical competent cells protocol	Tranformation efficiency kit
	o/n culture of E. coli BL21(DE3)	Chemical competent cells protocol	Tranformation efficiency kit	Colonies have the wrong color
Propagations	Transformed   AH001-AH008 into TOP10	AH001-AH004 & AH006	Stored in +4°C
		No colonies for   AH005, AH007 or AH008

Click here to read the full lab notes of week 1.

Week 2

Worked With	WorkFlow						May 25th - May 29th
Preparations Propagating	Transformed AH007-AH009, AH005 & AH019 into TOP10	AH007-AH009 & AH019	o/n cultures	MiniPrep
	o/n culture of  AH001-AH004 & AH006	MiniPrep
	Re-transform AH005 into TOP10	Plates in rt over weekend
Checking Competent Cells	o/n culture of BL21(DE3) transformants	MiniPrep	PCR with VF2 & VR	Smeared gels
			AH001+   AH003+AH008 -> AH013	Repeat ligation	Restrict & extract from gel	Transform into TOP10	Plates in rt over weekend
Testing RBS's Assembly	Restrict AH001-AH004 & AH006-AH008	Ligation	AH002+   AH004+AH008 -> AH014	Repeat ligation	Restrict & extract from gel	Transform into TOP10	Plates in rt over weekend
			AH001+    AH006+AH008 -> AH011	Repeat ligation	Restrict & extract from gel	Transform into TOP10	Plates in rt over weekend

Click here to read the full lab notes of week 2.

Week 3

Worked With	WorkFlow						June 1st - June 5th
Preparations Propagating	Transform     AH020 & AH021 into TOP10	Plates in rt  over weekend
Testing RBS's Assembly	No colonies for AH011 or AH014	re-assemble	Restrict & extract from gel	Ligate & tranform into TOP10	No colonies
	AH005 & AH013 OK	o/n cultures	MiniPrep
	Assembling AH015	Ligate AH002+   AH005+AH008	Restrict & extract from gel	Transform into TOP10	o/n cultures	No colonies on the plate

Click here to read the full lab notes of week 3.

Week 4

Worked With	WorkFlow						June 8th - June 12th
Preparations Propagating	o/n cultures for AH020 & AH021	MiniPrep
Testing RBS's Assembly	Re-assembling   AH011, AH013 & AH014	Transform into TOP10	o/n cultures	MinPrep
	Assembling AH015	Ligate AH002+   AH005+AH008	Restrict & extract from gel	Transform into TOP10	o/n cultures	MiniPrep

Click here to read the full lab notes of week 4.

Week 5

Worked With	WorkFlow						June 15th - June 19th
						AH016 wrong size on gel
				Colonies in BL21(DE3)	o/n cultures & MiniPrep	AH018 OK on gel
Testing RBS's Assembly	Screening AH011 & AH013-AH015 by gel electrophoresis	AH011, AH013 & AH015 OK	Assembling AH016 & AH018	Transform into TOP10 & BL21(DE3)	T7 promoter doesn't work in TOP10...
		AH014 incorrect size	Assembling AH017 with multiple AH014 plasmids	Restrict & extract from gel	Transform into BL21(DE3)	Colonies are not blue
	Re-assembling AH016 & AH018	Restrict extract from gel	Transform into BL21(DE3)	Colonies are not blue
Preparations Linearized Backbones	o/n cultures of AH007-AH009	MiniPrep	Restrict & extract from gel	Low concentration

Click here to read the full lab notes of week 5.

Week 6

Worked With	WorkFlow						June 22nd - June 26th
Preparations Linearized Backbones	o/n cultures of AH007-AH009	MiniPrep	Restrict & extract from gel
Testing RBS's Assembly	Re-assembling AH016 & AH018	Transform into BL21(DE3)	No colonies
Propagation	o/n cultures of  AH016 & AH018 in TOP10	MiniPrep	Transform into BL21(DE3)	Plates in rt over the weekend
Measurement	AH018 in BL21(DE3)	o/n culture	IPTG induction &     blue chromoprotein measurement	No blue chromoprotein
		Refresh on IPTG plate	No blue chromoprotein

Click here to read the full lab notes of week 6.
Blue chromoprotein measurement data from 23.6. can be found here.

Week 7

Worked With	WorkFlow						June 29th - July 3rd
Preparations Linearized Backbones	Transform AH007-AH009 into TOP10	No colonies (mistake in protocol)	Use iGEM's linearized backbones
Testing RBS's	o/n cultures of   AH016&AH018 in BL21	IPTG induction	No Blue chromoprotein
	Refresh AH016 & AH018 on IPTG plate	No blue chromoprotein
Violacein Plasmid Construction	PCR AH020 with P012&P013	PCR purification	VioA waits for 2nd PCR
	PCR AH20 with P024&P025	PCR unsuccesful
	PCR AH20 with P034&P035	PCR purification	VioE waits for 2nd PCR

Click here to read the full lab notes of week 7.
Blue chromoprotein measurement data from 30.6. can be found here.

Week 8

Worked With	WorkFlow						July 6th - July 10th
Preparations Competent Cells	Preparing   PK BL21(DE3) competent cells	(Cells called PK's BL21(DE3))	Cells stored in -80°C
Cellulose Plasmid Construction	PCR AH021 with P019&P020	Problem with PCR machine	Re-do PCR	PCR purifcation	BglX waits for 2nd PCR
	AH029 into TOP10	Transformants stored in +4°C
Testing RBS's	Transform AH016 & AH018 into PK BL21(DE3)	Refresh transformants on IPTG plates	No blue chromoprotein
Violacein Plasmid Construction	Gradient PCR for AH020 with P024&P025	PCR purification

Click here to read the full lab notes of week 8.

Week 9

Worked With	WorkFlow						July 13th - July 17th
Preparations Propagations	o/n culture of AH029	MiniPrep
		CAR2 OK	PCR purification	Low yield
Propane Plasmid 1 Construction	Construct from 3 parts: CAR1-3	PCR for CAR1-3	CAR1 & 3 unspecific bands	Gradient PCR for CAR3	PCR with DMSO for CAR3	Store in -20°C
			PCR with DMSO for CAR1	No product
		PCR with DMSO for AtoB1	Store in -20°C
Propane Plasmid 2 Construction	Construct from 4 parts: AtoB1-4	PCR for AtoB 1-4	Gradient PCR for AtoB2	PCR with DMSO for AtoB2	Store in -20°C
		Gradient PCR for AtoB3	Extract & purify from gel	Store in -20°C
	Extract & purify AtoB4 from gel	Store in -20°C
GFP Brick Construction	o/n culture of AH044	MiniPrep

Click here to read the full lab notes of week 9.

Week 10

Worked With	WorkFlow						July 20th - July 24th
Preparations Linearized Backbones	Transform AH043 into TOP10	o/n culture	MiniPrep	Restrict
Propane Plasmid 1 Construction	Gradient PCR for CAR1	Extract & purify from gel	Low yield	TOPO Blunt End cloning and transform into TOP10t	Screen with colony PCR	Screen more colonies
	CAR2 PCR ran on gel	Gradient PCR	Extract & purify from gel	Illegal restriction site found in the part
	CAR3 PCR with DMSO run on gel	Extract purify from gel unclean product	TOPO Blunt End cloning and transform into TOP10t	Screen with colony PCR	Extract & purify from gel
Propane Plasmid 2 Construction	Run AtoB1 PCR with DMSO on gel	Extract & purify from gel	Low yield
	Run AtoB2 PCR with DMSO on gel	Extract & purify from gel	Low yield

Click here to read the full lab notes of week 10.

Week 11

Worked With	WorkFlow						July 27th - July 31st
Preparations Linearized Backbones	Re-restrict AH043	Gel purification	Low yield	Re-restrict
Propagations	o/n culture of AH031	MiniPrep
	Transform AH033 into TOP10	o/n culture	MiniPrep	Low yield	o/n culture	MiniPrep	Low yield, but stored in -20°C
Propane Plasmid 1 Construction	Continue CAR1 colony screening	Screen more colonies	PCR purification
	PCR for new CAR2	Gradient PCR with KAPA polymerase	PCR purification
	Re-do colony PCR for CAR3 successful TOPO colony	Re-do colony PCR
		o/n culture of the colony	MiniPrep	PCR
	KAPA gradient PCR for CAR3	Gel purification
Propane Plasmid 2 Construction	TOPO Blunt End Cloning for AtoB1 and transformation into TOP10	Screening with colony PCR	PCR purification
	KAPA gradient PCR for AtoB3&4	PCR purification for AtoB3
		Gel purification for AtoB4
Cellulose Plasmid Construction	2nd PCR for BglX (from AH021)	Stored in -4°C over the weekend
	PCR for Cen1 & Cen2	Gradient PCR with KAPA polymerase	PCR purification for Cen2
			Gel purification for Cen1
Violacein Plasmid Construction	2nd PCR for VioA (from AH020)	Stored in -4°C over the weekend

Click here to read the full lab notes of week 2.

Week 12

Worked With	WorkFlow						August 3rd - August 8th
Preparations Linearized Backbones	Check restricted AH043 on gel	Use for CAR OE-PCR
	Re-restrict with high amount of DNA	Extract & purify from gel	Low yield
	New o/n culture of AH043 (needed more backbone)	MiniPrep	Remove insert	Use for CAR NEBuilder
Fixing Commercial Backbones	Restrict and ligate AH006 into AH031&AH033	Transform into TOP10
Propane Plasmid 1 Construction	OE-PCR without backbone	Weak product band	PCR purification	Gradient PCR	No product
	OE-PCR with backbone	Transform into TOP10	Red colonies
	NEBuilder protocol	Transform into TOP10	Screen with colony PCR	o/n culture	MiniPrep	Send for sequencing
Violacein Plasmid Construction	PCR purification for VioA 2nd PCR	Stored in -20°C
Cellulose Plasmid Construction	PCR purification for VioA 2nd PCR	Stored in -20°C
Amph protein	Gradient PCR P044&P045, P046&P051, P045&P046	Extract & purify from gel P044&P045	DNA not clean	Store in -20°C
		Stored P046&P051 and P045&P046 product gels to +4°C
GFP protein	Gradient PCR with P028&P029	PCR purification
Propane Measurement	o/n culture of Pauli's propane strain	Propane measurement	No propane detected

Click here to read the full lab notes of week 2.

Week 13

Worked With	WorkFlow						August 10th - August 14th
Amphiphilic Protein Construction	PCR amph with P044&P045	No product	Gradient PCR	No product	Continue with last week's product
Preparations Backbones	Restrict AH043	Stored in -20°C
GFP & Amph Bricks Assembly	Restrict & ligate GFP and Amph into AH009	Transform into TOP10	Screen with colony PCR	o/n culture	MiniPrep	Check on gel	Send for sequencing
Propane Plasmid 2 Assembly	NEBuilder protocol	Transform into TOP10	Screen with colony PCR	Re-do NEBuilder and transfrom to TOP10	Screen with colony PCR	o/n culture of one potential colony	o/n culture turned red
	ELIC protocol	Transform into TOP10	No colonies
Cellulose Plasmid Assembly	NEBuilder protocol and transform into TOP10	Screen with colony PCR	Mistake in protocol	Re-do NEBuilder and transform into TOP10	Screen with colony PCR	o/n cultures and MiniPrep of white colonies	Restrict & check on gel
	ELIC protocol	Transform into TOP10	Screen with colony PCR	Mistake in protocol	Re-do ELIC and transform into TOP10	Screen with colony PCR
GFP Brick Validation	Restrict AH013, AH015 & AH007	Ligate AH007, AH013 & GFP	Transform into TOP10	RT over weekend
		Ligate AH007, AH015 & GFP	Transform into TOP10	RT over weekend

Click here to read the full lab notes of week 2.

Week 14

Worked With	WorkFlow						August 17th - August 21st
Preparations Propagations	Transform AH045 into TOP10	o/n culture	MiniPrep
Propane Plasmid 1 Assembly	Restrict & ligare CAR into AH009	Transform into TOP10	o/n culture	MiniPrep	Restrict & check on gel
	Sequencing of Propane Plasmid 1 unsuccessful	Send construct in AH009 for sequencing
Validation	Transform into Pauli's BL21(DE3)
Amphiphilic Propane 1	Gradient PCR for CAR amph parts	CAR amph3 purified from gel
	PCR piurification for CAR amph4
Propane Plasmid 2 Construction	OE-PCR	No product
	Check old NEBuilders on gel	Re-transform into TOP10	o/n cultures of colorless colonies	MiniPrep	Restrict & check on gel
Cellulose Plasmid Construction	OE-PCR	Extract & purify from gel	Concentration too low
		PCR purification	NEBuilder with AH009	Transform into NEB5alpha over weekend
	Check old NEBuilder on gel	Re-transform into TOP10	o/n of colorless colonies	MiniPrep	Restrict & check on gel
GFP Assembly	2A assembly for AH013/AH015, GFP & pSB1C3	Transform into TOP10	Colonies can’t be separated	3A assembly into pSB1T3	Transform into TOP10	No colonies
	Propagate GFP in AH009	MiniPrep	Send to Slovenia

Click here to read the full lab notes of week 2.

Week 15

Worked With	WorkFlow						August 24th - August 28th
Propane Plasmid 1 Validation	Prepare SDS-PAGE samples	Unclear results
	Analytical restriction	Wrong sized bands
	Prepared cells for purpald test	Stored in -70°C
Propane Plasmid 2 Construction	Repeat NEBuilder	Transform into TOP10
	Repeat NEBuilder with AH009	Transform into TOP10	Colony PCR
GFP Biobrick Assembly	3A Assemble GFP + AH045 in pSB1K3	Transform into TOP10	O/n cultues	MiniPrep	Restrict & Check on gel
	2A Assembly GFP + AH045 in pSB1K3	Transform into TOP10	Store in +4 °C
GFP+Amph Assembly	3A Assemble AH045-GFP + amphlinker in pSB1A3	Transform into TOP10 over weekend
Cellulose Plasmid Construction	Repeat NEBuilder	Transform into TOP10
	Repeat NEBuilder with AH009	Transform into TOP10	Colony PCR
Amphiphilic Propane1	NEBuilder	Transform into TOP10	Colony PCR
		O/n culture	MiniPrep

Click here to read the full lab notes of week 2.

Week 16

Worked With	WorkFlow						August 31st - September 4th
Propane Plasmid 1 Validation	New SDS-PAGE	Unclear results
Propane Plasmid 2 Construction	o/n culture of NEBuilder colonies	MiniPrep	Restrict & check on gel	No insert	Screen more colonies with colony PCR
Cellulose Plasmid Construction	o/n culture of NEBuilder colonies	MiniPrep	Restrict & check on gel	No insert	Screen more colonies with colony PCR
Amph Propane 1 Construction	Restrict & check on gel	Re-do NEBuilder	Transform into NEB5alpha	O/n cultures	MiniPrep
GFP Amph Assembly	O/n cultures	MiniPrep	Restrict & check on gel	O/n cultures of more colonies	MiniPrep, restrict & check on gel	Prepare samples for microscopy	Problem with imaging
Amphiphilic Brick Assembly	PCR purification	3A assemble AH045 + Amph in pSB1C3	Transform into TOP10	O/n cultures & MiniPrep	Restrict & Check on gel	More o/n cultures & MiniPrep	Restrict & Check on gel, results unclear

Click here to read the full lab notes of week 2.

Week 17

Worked With	WorkFlow						September 7th - September 11th
Propane Plasmid 1 Validation	Colony PCR with P001 & VR	Results next week
Propane Plasmid 2 Construction	o/n culture of NEBuilder colonies	MiniPrep	Restrict & check on gel	AtoB maybe OK	Restrict & check on gel again	Unclear results	Colony PCR, results next week
Cellulose Plasmid Construction	o/n culture of NEBuilder colonies	MiniPrep	Restrict & check on gel	No insert	Due to the time constraint, the construction is not repeated
Amph Propane 1 Construction	Restrict & check on gel	Incorrect size	Due to the time constraint, the construction is not repeated
GFP Amph Assembly	Restrict & check on gel	Incorrect size	Re-assemble	Transform into TOP10	GFP not expressed	Transform old ligation mix into TOP10	GFP not expressed
Validation	Imaged with fluorescent microscope
Amphiphilic Brick Assembly	Restrict & check on gel	Incorrect size	Re-assemble	Transform into TOP10	O/n cultures	MiniPrep

Click here to read the full lab notes of week 2.