PCR pDawn BioBricks

PCR YC155, pDawn I, II and III

Aim: first step for pDawn amplification and mutagenesis

Mix PCR preparation:

	YC155	pDawn I	pDawn II	pDawn III
MQ water	39,5 µL	39,5 µL	39,5 µL	39,5 µL
Mg2+ 50 mM	1,5 µL	1,5 µL	1,5 µL	1,5 µL
Buffer RB	5 µL	5 µL	5 µL	5 µL
dNTPs 10µL	1 µL	1 µL	1 µL	1 µL
Primer Fwd 50µM	1µL YC155 Fwd	1µL pDawn Fwd I	1µL pDawn Fwd II	1 µL pDawn Fwd III
Primer Rev 50µM	1µL YC155 Rev	1µL pDawn Rev I	1µL pDawn Rev II	1 µL pDawn Rev III
DNA	1µL PCR YC155	1µL pDawn	1µL pDawn	1 µL pDawn
Taq Pol	0.5 µL	0.5 µL	0.5 µL	0.5 µL

iGEMTAQ program

Electrophoresis

Aim: Migration of PCR product

Gel : 1,2 g agarose + 120 ml of TAE 1x + 150 µl of BET
Migration of PCR products YC155, pDawn I, II and III

We get expected bands for YC155, pDawn I and II but there is no amplification for pDawn III
First explanation: human error We should make the reaction again

Gel Purification

Aim: purification of fragment from PCR

QIAquick gel extraction
2 bands of each fragment into 1 column
Concentrate into 50 µl of Elution Buffer

PCR YC155, pDawn I, II and III

Mix PCR preparation:

	pDawn III
MQ water	39,5 µL
Mg2+ 50 mM	1,5 µL
Buffer RB	5 µL
dNTPs 10µL	1 µL
Primer Fwd 50µM	1µL pDawn Fwd III
Primer Rev 50µM	1µL pDawn Rev III
DNA	1µL pDawn
Taq Pol	0.5 µL

iGEMTAQ program

Electrophoresis

Aim: Migration of PCR product

Gel : 1,2 g agarose + 120 ml of TAE 1x + 150 µl of BET
Migration of PCR products purified YC155 and pDawn III

No amplification of pDawn III
YC155 quantification = 128 ng.µL^-1

30 June 15

Digestion of pSB1C3

Tube preparation:

Tube	pSB1C3 I	pSB1C3 II	pSB1C3 III
Water	3 µL	3 µL	8 µL
Buffer 2.1	5 µL	5 µL	5 µL
Plasmid pSB1C3	40 µL	40 µL	35 µL
Enzyme EcoRI	1 µL	1 µL	1 µL
Enzyme PstI	1 µL	1 µL	1 µL

NEB double digestion
Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 65min

Electrophoresis

Aim: check for digestion

Migration of the three digested pSB1C3 samples

Gel Purification

QIAquick gel extraction
4 bands (1,175 g) into 1 column
Concentrate into 30 µL of Elution Buffer

Electrophoresis

Aim: quantification of DNA

Migration of digested pSB1C3 samples

Quantification by Image J: 91 ng.µl^-1

12 June 15

Result from 10.06.15

Culture liquid

Miniprep

Protocol from MOBIO Kit

• pSB1C3: 20 ml into 4 columns, eluted with 30 µl (pooled together)

Electrophoresis

Gel: 1,2 g of agarose + 120 ml of TAE 1x + 150 µl of BET
Migration of purified pSB1C3

Quantification of pSB1C3 by Image J = 35 – 40 ng.µL^-1
4 µg (min) of pSB1C3 are requiring for digestion of the plasmid to make a Gibson reaction

• 35 ng x 115 µl (remaining) = 4,025 µg all the remaining 115 µL of pSB1C3 should be digested

11 June 15

Miniprep

Protocol from QIAgen, UltraClean® Standard Mini plasmid Prep Kit

• BBa_I712074
• BBa_K124003

Glycerol stock

Stock of BBa_I712074 and BBa_K124003 (cf protocol for glycerol stock), -80°C

Electrophoresis

Migration of purified BBa_I712074 and BBa_K124003

Bands do not correspond exactly to expected weights (supercoiled)

Plate preparation

3 plates: 20 mL of LB agar + 20 µL of Cam

Liquid culture

Double click tube: 3 mL of LB broth + 3 µL of antibiotic

Tube preparation

Tube	Antibiotic
BBa_I712074	Cam
BBa_K124003	Cam
Control Negative	Cam

Survival checking of bacteria after a night spent into a -20°C freezer
Erlenmeyer: culture of bacteria transformed with pSB1C3 coming from glycerol stock; 20 mL of LB broth + 20 µL of Cam

10 June 15

Amplification of iGEM parts

Amplification of bacteria

Amplification of bacteria already transformed with BBa_I712074 (Promoter T7) or BBa_K124003 ( Endolysin/Holin)
On petri dish: 20 mL LB Agar + 20µL Amp & 20µL Kan
LB Broth: 2,5 mL of LB + 2,5µL of Amp & 2,5µL of Kan

9 June 15

Miniprep: QIAgen vs MOBIO

Miniprep test using liquid culture of Terminator T7 and RBS to check the efficiency of miniprep kit from QIAgen and MOBIO

Electrophoresis

Aim: Verification of the purification

Migration of miniprep products

Bands have more migrated than expected but it could be due to the folding of circular plasmid. Both kits are efficient.

Plasmid digestion

Tube preparation:

Tube	Terminator T7	RBS
Water	12 µL	12 µL
Buffer 2.1	2 µL	2 µL
Plasmid pSB1C3	5 µL	5 µL
Enzyme EcoRI	0.5 µL	0.5 µL
Enzyme PstI	0.5 µL	0.5 µL

NEB double digestion
Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 1h10
Storage: -20°C

Electrophoresis

Migration of digested T7 and bJun from 29.05.2015 and digested T7 and RBS from 05.06.2015

After digestion we get expected band for T7 terminator and RBS from minipreps of the day

Gibson Assembly

Aim: first step of our VVD Biobrick

Master Mix from University of Freiburg (15µL)
Mix of DNA fragment:

• 1.5 µL pSB1C3
• 1 µL PCR.2 VVD1 (diluted 1/100)
• 2 µL PCR.2 VVD2 (diluted 1/100)
• 1 µL YC155 (diluted 1/10)

5 µL of the DNA mix is adding to the Master mix
Put immediately into a hot bath at 50°C for 1 hour

Transformation

Aim: transformation of E.Coli with Gibson Assembly product

20 µL of competent cells + 5 µL of Gibson product
Protocol for bacteria transformation: using 200 µl of LB broth before incubation for 1 h at 37°C and discarding 150 µl of supernatant before plating

5 June 15

Plasmid digestion (pSB1C3)

Aim: Extraction of the biobrick’s backbone for Gibson Assembly

Into aliquots, added plasmids or nothing (control) as following:

Tube	pSBC3
Buffer 2.1	2 µL
Plasmid pSB1C3	17 µL
Enzyme EcoRI	0.5 µL
Enzyme PstI	0.5 µL

NEB double digestion Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 1h10
Storage: -20°C

Electrophoresis

Aim: electrophoresis for gel purification

Migration of digested pSB1C3, VVD1 PCR2, VVD2 PCR2, YC155, YN155 I PCR2, YN155 II PCR2

We get expected bands
Extraction of the heaviest band of digested pSB1C3 and all the others expected bands for VVD 1, VVD 2, YC155, YN155 1, YN155 2.

Gel Purification

QIAquick gel extraction

VVD 1	180 mg				VVD 2	590 mg
YN155 1	180 mg				YN155 2	200 mg
YC155	120 mg				pSB1C3	190 mg

Concentrate into 30 µl of Elution Buffer Concentrate into 30 µl of Elution Buffer

Electrophoresis for quantification

Aim: quantification of ADN to perform Gibson Assembly

Migration of digested pSB1C3, VVD1 PCR2, VVD2 PCR2, YC155, YN155 I PCR2, YN155 II PCR2

There is not enough DNA to detect and quantify pSB1C3 and YC155 Try again

Electrophoresis for quantification - 2^nd

Migration of digested pSB1C3, VVD1 PCR2, VVD2 PCR2, YC155, YN155 I PCR2, YN155 II PCR2

Quantification of DNA fragments by Image J:

Fragments	Concentration (ng.µL-1)	Lenght	Molecular weight (g.mol-1)	Molarity (mol.L-1)	1/ratio	Dilution factor	Molarity after dilution
pSB1C3	0.92	2 200	1 430 000	0.000000000642	177.7	1	0.000000000642
VVD1	15.71	250	162 500	0.000000096698	1.18	151	0.000000000642
VVD2	7.65	250	162 500	0.000000047103	2.42	73	0.000000000642
YC155	2.50	400	260 000	0.000000009631	11.85	15	0.000000000642
YN155.1	8.42	200	130 000	0.000000064805	1.76	101	0.000000000642
YN155.2	14.84	200	130 000	0.000000114117	1	178	0.000000000642

Liquid culture

Double click tube: 3 mL of LB broth + 3 µL of antibiotic

Plasmid	Antibiotic				Plasmid	Antibiotic
Terminator T7	Cam				bFos	Amp
RBS	Cam				bJun	Amp
Control Negative	Cam				Control Negative	Amp

Concentrate into 30 µl of Elution Buffer Concentrate into 30 µl of Elution Buffer

4 June 15

Results from 29.05.2015

Results of transformation with Gibson assembly product

No bacterial growth

Electrophoresis of digestion product

Aim: check for digestion product from 29.05.2015

No bands for miniprep samples or for digestion products
The miniprep has failed

2 June 15

Liquid culture results

Results of the liquid culture from 28/05/2015

After 24h of growth:
Terminator T7 bacteria growth strongly into both tubes
bJun bacteria growth into strongly one tube only, the other one is “cloudy”
bFos both tube are “cloudy”
Control negative clear

Glycerol stock

Stock of Terminator T7 and bJun (cf protocol for glycerol stock), -80°C

Miniprep

Aim: plasmid purification from transformed bacteria

Protocol from QIAgen, UltraClean® Standard
Mini plasmid Prep Kit
Terminator T7: 2 tubes
bJun: 1 tube

Plasmid digestion (Terminator T7 & bJun)

Terminator T7

Tube	Terminator T7	Control negative
Water	12 µL	13 µL
Buffer 2.1	2 µL	2 µL
DNA	5 µL	5 µL
Enzyme EcoRI	0.5 µL	--
Enzyme SpeI	0.5 µL	--

NEB double digestion Buffer 2.1:

• EcoRI: 100%
• SpeI: 100%

bJun

Tube	bJun	Control negative
Water	12 µL	13 µL
Buffer 2.1	2 µL	2 µL
DNA	5 µL	5 µL
Enzyme EcoRI	0.5 µL	--
Enzyme PstI	0.5 µL	--

NEB double digestion Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 1h10
Storage: -20°C

Gel Purification

Aim: purification of fragment from 28.05.2015

Use of a QIAquick gel extraction kit with the protocol of QIAgen
Gel weight = 270 mg

Gibson Assembly

Aim: first step of our VVD Biobrick

Master Mix from University of Freiburg (15µL)
Mix of DNA fragment:

• 1 µL pSB1C3
• 2 µL PCR.2 VVD1
• 2 µL PCR.2 VVD2
• 2 µL YC155

5 µL of the DNA mix is adding to the Master mix
Put immediately into a hot bath at 50°C for 1 hour

Transformation

Aim: transformation of E.Coli with Gibson Assembly product

Transformation

Plasmid	Plate
3 µL of plasmid	20 µL on plate
	50 µL on plate
6 µL of plasmid	20 µL on plate
	50 µL on plate
Control negative	100 µL on plate

Incubate at 25°C during 60 hours

29 May 15

Amplification of pSB1A2 (part BBa_J61100) into E.coli DH5 alpha

Plate preparation

Aim: preparation of selective LB agar plate

Add 40 ml of LB agar into a Falcon tube (50ml) + 40µl antibiotic For 20 ml / plate (2 plates)

Agar plates preparation

Plasmid	Antibiotic	Number of plate	Remarks
pSB1A2
	Ampicillin	1	50 µl of transformed cells
		1	20 µl of transformed cells
Control negative
	Amplicilin	1	50 µl of transformed cells

Antibiotics concentration:

• Ampicilin: 50 µg.ml^-1 diluted 1/1000

Cell transformation

E.coli DH5 alpha (NEB5 alpha competent coli)
Stock: 6 tubes 0.20 ml / tube; 50 pg.µl^-1
Aliquot of 50 µl of competent E.coli / transformation
100 µl remaining competent cells have been put at -80°C again (loss of efficiency)

Into aliquots, added plasmids or nothing (control) as following:

Tube n°	Reagent added	Volume (µl)
1	pSB1A2	1
2	Control negative	--

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min, do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 150 µl
Plate bacteria following the table about plate preparation
Incubate at 37°C, O/N

Expected results:
No colonies into the control plate
Many colonies into the other plate

Plasmid digestion (pSB1C3)

Aim: Extraction of the biobrick’s backbone for Gibson Assembly

Into aliquots, added plasmids or nothing (control) as following:

Tube	pSBC3
Water	16 µL
Buffer 2.1	2 µL
Plasmid pSB1C3	1 µL
Enzyme EcoRI	0.5 µL
Enzyme PstI	0.5 µL

NEB double digestion Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 1h10
Storage: -20°C

Electrophoresis

Aim: check for digestion

1 gel for purification: 110ml TAE 1X + 1,1g agarose + 140µl BET
Sample (purification): 20µl of digestion product+ 2µl of loading dye (6x)
Sample (quantification): 1µl of DNA + 2 µl of loading dye (6x) + 9µl of water MQ
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl loading dye (6x) + 9 µl water MQ
110 V, 45 min

Liquid culture

Antibiotics concentration:
Tube 1 & 2: 3 mL LB + 3 µL Cam; colonies from “1µL” plate of Terminator T7 transformation
Tube 3 & 4: 3 mL LB + 3 µL Amp; colonies from “50µL” plate of bFos
Tube 5 & 6: 3 mL LB + 3 µL Amp; colonies from “50µL” plate of bJun
Tube 7 & 8: 3 mL LB
37°C, 120 rpm, O/N

28 May 15

Results from cell tranformation

Results of the transformation from 25/05/2015:

After 24h of growth:
No colonies into negative controls Many colonies appears into plates with bFos and bJun transformed bacteria No colonies into plates with pSB1A2 transformed bacteria:

• It could be due to the presence of 2 antibiotics (high environmental pressure)

26 May 15

RBS BioBricks

Recover dried DNA sent by iGEM (part BBa_J61100)

Plasmid characteristic:

• Part name: BBa_J61100
• Plasmid: pSB1A2
• Part name: BBa_J61100
• Resistance: AK (Amplicilin/Kanamicin)

Punch a hole without removing the foil
Pipette 10µl of water MQ (up and down)
Let sit for 5 minutes to make sure the dried DNA is fully resuspended

Recovered plasmid have been put into a 50µl eppendorf tube called “BBa_J61100, RBS”

Amplification of bFos, bJun, pSB1A2 (part BBa_J61100) into E.coli DH5 alpha

Plate preparation

Aim: preparation of selective LB agar plate

Add 40 ml of LB agar into a Falcon tube (50ml) + 40µl antibiotic For 20 ml / plate (2 plates)

Agar plates preparation

Plasmid	Antibiotic	Number of plate	Remarks
bFos
	Amplicillin	1	50 µl of transformed cells
		1	100 µl of transformed cells
bJun
	Amplicillin	1	50 µl of transformed cells
		1	100 µl of transformed cells
pSB1A2
	Ampicillin + Kanamycin	1	50 µl of transformed cells
		1	100 µl of transformed cells
Control negative
	Amplicilin	1	50 µl of transformed cells
	Kanamycin	1	50 µl of transformed cells

Antibiotics concentration:

• Ampicilin: 50 µg.ml^-1 diluted 1/1000
• Kanamycin: 50 µg.ml^-1 diluted 1/1000

Cell transformation

E.coli DH5 alpha (NEB5 alpha competent coli)
Stock: 6 tubes 0.20 ml / tube; 50 pg.µl^-1
Aliquot of 50 µl of competent E.coli / transformation

Into aliquots, added plasmids or nothing (control) as following:

Tube n°	Reagent added	Volume (µl)
1	bFos	1
2	bJun	1
3	pSB1A2	1
4	Control negative	--

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min, do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 150 µl
Plate bacteria following the table about plate preparation
Incubate at 37°C, O/N

Expected results:
No colonies into the 2 control plates
Many colonies into all the other plates
Colonies from pSB1A2 transformation should exhibit red color

25 May 15

PCR VVD BioBricks

PCR.2 Nter (YN155)

Aim: second step of mutagenesis for YN155

PCR.2 YN155 preparation

Component	PCR.2 YN155 I (x2)	PCR.2 YN155 II (x2)	Control - YN155 I (x2)	Control - YN155 II
Primer Fwd YN155 1	1 µl	--	--	--
Primer Rev YN155 3	1 µl	--	--	--
Primer Fwd YN155 4	--	1 µl	--	--
Primer Rev YN155 2	--	1 µl	--	--
PCR.1, YN155 I	1 µl	--	1 µl	--
PCR.1, YN155 II	--	1 µl	--	1 µl
Taq Pol	0.5 µl	0.5 µl	0.5 µl	0.5 µl

Mix PCR (x8) without primers, DNA and Taq pol
PCR cycle IGEM TAQ program, Tm = 61,3°C

Electrophoresis

Aim: check for digestion

Remaining gel (120ml TAE 1X + 1,2g agarose + 150µl BET) with 8 wells
Sample: 10 µl of PCR product+ 2 µl of loading dye (6x) only 10 µl loaded.
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl of loading dye (6x) + 9 µl water MQ only 10 µl loaded.
110 V, 26 min

We get expected bands with a good intensity meaning DNA have been highly amplified.

Purification of PRC product

Aim: purify and concentrate amplified DNA

Use of a QIAquick PCR Purification kit and using a microcentrifuge.

For step 1: add 5 volumes of PB buffer for 1 volume of PCR product

• 200 µl of PB buffer added into both coupled of tube with 40 µl of remaining PCR product
• Mix of same PCR product together into a new tube
• Put mixes into their own purification column

Then, we followed the protocol from the supplier

At the end of the purification, we get 2 tubes: “PCR2, YN I, 22/05/2015”, “PCR2, YN II, 22/05/2015”
Stored at -20°C.

Liquid culture

Use of 3 colonies (3 tubes) from pSB1C3 (BBA_B0015) 1µl and pSB1C3 (BBA_B0015) 4µl
3ml LB Broth + 3µl Cam in double click tubes (12ml) + 1 control tube (3ml LB Broth)
37°C, 120rpm, O/N

22 May 15

PCR VVD BioBricks

PCR.1 YFP Cter (YC155) & Nter (YN155)

Aim: amplification of YC155 and first step of mutagenesis for YN155

PCR YC155 and YN155 preparation

Component	PCR YC155 (x2)	PCR.1 YN155 I (x2)	PCR.1 YN155 II (x2)	Control - YC155	Control - YN155
Primer Fwd YC155	1 µl	--	--	--	--
Primer Rev YC155	1 µl	--	--	--	--
Primer Fwd YN155 I	--	1 µl	--	--	--
Primer Rev YN155 I	--	1 µl	--	--	--
Primer Fwd YN155 II	--	--	1 µl	--	--
Primer Rev YN155 II	--	--	1 µl	--	--
Plasmid pBiFC-bFos	1 µl	--	--	1 µl	--
Plasmid pBiFC-bJun	1 µl	--	--	1 µl	--
Taq Pol	0.5 µl	0.5 µl	0.5 µl	0.5 µl	0.5 µl

Mix dNTPs: 2 µl of each dNTP
Mix PCR without primers, plasmids and Taq Pol
PCR cycle IGEM TAQ program

Electrophoresis

Aim: check for digestion

1 gel: 120ml TAE 1X + 1,2g agarose + 150µl BET
Sample: 10 µl of PCR product+ 2 µl of loading dye (6x) only 10 µl loaded.
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl of loading dye (6x) + 9 µl water MQ only 10 µl loaded.
110 V, 25 min

We get expected bands with a good intensity meaning DNA have been highly amplified

Purification of PRC product

Aim: purify and concentrate amplified DNA

Use of a QIAquick PCR Purification kit and using a microcentrifuge.

For step 1: add 5 volumes of PB buffer for 1 volume of PCR product

• 200 µl of PB buffer added into both coupled of tube with 40 µl of remaining PCR product
• Mix of same PCR product together into a new tube
• Put mixes into their own purification column

Then, we followed the protocol from the supplier

At the end of the purification, we get 3 tubes: “PCR1, YC155, 21/05/2015”, “PCR1, YN155 I, 21/05/2015”, “PCR1, YN155 II, 21/05/2015”
Stored at -20°C.

Backbone & Terminator

Transformation pSB1C3 – double T7 terminator (BBa_B0015)

Aim: plasmid amplification

3 plates with 25 ml LB medium + 25 µl of Cam antibiotic / plate
Recover dried DNA sent by iGEM

• Punch a hole without removing the foil (Plate 3, well F3)
• Pipette 10 µl of water MQ (up and down)
• Pipette 10 µl of water MQ (up and down)
• Protocol for bacteria transformation

Cell transformation

• E.coli DH5 alpha (NEB5 alpha competent coli)
• Aliquot of 50 µl of competent E.coli / transformation

Into aliquots, added plasmids or nothing (control) as following:

Tube n°	Reagent added	Volume (µl)
1	pSB1C3 – double T7 terminator (BBa_B0015)	1
2	pSB1C3 – double T7 terminator (BBa_B0015)	4
3	Control negative	--

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min. Do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 100 µl
Plate bacteria and incubate at 37°C, O/N

Results
Control was cloudy as cultures (lack of antibiotics) should be done again
21 May 15

PCR VVD BioBricks

PCR.2 VVD - 2nd part

Aim: second step for VVD amplification and mutagenesis

End of PCR.2 VVD preparation from 07.05.15

Component	PCR.2 VVD 1 (x2)	PCR.2 VVD 2 (x2)	Control negative	Control negative
DNA Mix "PCR.1 VVD1"	1 µl	--	1 µl	--
DNA Mix "PCR.1 VVD2"	--	1 µl	--	1 µl
Taq Pol	0.5 µl	0.5 µl	0.5 µl	0.5 µl

Run of the IGEM TAQ program

Electrophoresis

Aim: check for digestion

1 gel: 120ml TAE 1X + 1,2g agarose + 160µl BET
Sample: 10 µl of PCR product+ 2 µl of loading dye (6x) only 10 µl loaded.
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl loading dye (6x) + 9 µl water MQ only 10 µl loaded.
110 V, 30 min

We get expected bands about 250 bp meaning DNA have been highly amplified

Purification of PRC product

Aim: purify and concentrate amplified DNA

Use of a QIAquick PCR Purification kit and using a microcentrifuge.

For step 1: add 5 volumes of PB buffer for 1 volume of PCR product

• 200 µl of PB buffer added into both coupled of tube with 40 µl of remaining PCR product
• Mix of same PCR product together into a new tube
• Put mixes into their own purification column

Then, we followed the protocol from the supplier

At the end of the purification, we get 1 tube “PCR2, VVD1, 13/05/2015” and another one “PCR2, VVD2, 13/05/2015”
Stored at -20°C.

13 May 15

PCR VVD BioBricks

PCR.1 VVD - 2nd part

Aim: first step for VVD amplification and mutagenesis

PCR.1 VVD preparation

Component	PCR.1 VVD 1 (x2)	PCR.1 VVD 2 (x2)
PCR mix	43,5 µl	43,5 µl
Primer VVD Fwd 1	1 µl	--
Primer VVD Rev 1	1 µl	--
Primer VVD Fwd 2	--	1 µl
Primer VVD Rev 2	--	1 µl
Water	2 µl	2 µl
Plasmid VVD	1 µl	1 µl
Taq Pol	0.5 µl	0.5 µl

Run of the IGEM TAQ program

Electrophoresis

Aim: check for DNA amplification

Low melting point gel
Sample: 10 µl of PCR product+ 2 µl of loading dye (6x)
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl of loading dye (6x) + 9 µl water MQ
110 V, 26 min

We get expected bands about 200 bp with a good intensity meaning DNA have been highly amplified without contamination.

Purification of PRC product

Aim: purify and concentrate amplified DNA

Use of a QIAquick PCR Purification kit and using a microcentrifuge.

For step 1: add 5 volumes of PB buffer for 1 volume of PCR product

• 200 µl of PB buffer added into both coupled of tube with 40 µl of remaining PCR product
• Mix of same PCR product together into a new tube
• Put mixes into their own purification column

Then, we followed the protocol from the supplier

At the end of the purification, we get 1 tube “PCR1, VVD1, 07/05/2015” and another one “PCR1, VVD2, 07/05/2015”
Stored at -20°C.

PCR.2 VVD

Aim: second step for VVD amplification and mutagenesis

dNTP preparation (4µL):
1 µl dATP 100 mM
1 µl dCTP 100 mM
1 µl dTTP 100 mM
1 µl dGTP 100 mM
Store at -20°C

PCR.2 VVD preparation

Component	PCR.2 VVD 1 (x2)	PCR.2 VVD 2 (x2)	Control negative (x2)
Primer VVD Fwd 1	1 µl	--	--
Primer VVD Rev 3	1 µl	--	--
Primer VVD Fwd 4	--	1 µl	--
Primer VVD Rev 2	--	1 µl	--
Water	39.5 µl	39.5 µl	41.5 µl
Buffer RB	5 µl	5 µl	5 µl
dNTPs	0.5 µl	0.5 µl	0.5 µl
Mg2+ 50mM	1.5 µl	1.5 µl	1.5 µl

7 May 15

PCR VVD BioBricks

PCR.1 VVD

Aim: first step for VVD amplification and mutagenesis

dNTP preparation (4µL):
1 µl dATP 100 mM
1 µl dCTP 100 mM
1 µl dTTP 100 mM
1 µl dGTP 100 mM
Store at -20°C

PCR cycle – IGEM TAQ

	T°C	Time	Cycle
Initial denaturation	95	30 sec	1
Denaturation	95	30 sec	17
Annealing	60	30 sec
Extension	72	1 min / kb
Final extension	72	5 min	1
Hold	4	Infinite

Mix PCR preparation without primers, plasmid and Taq

MQ water	182,5 µl
Mg2+ 50 mM	7,5 µl
Buffer RB	25 µl
dNTPs	2,5 µl

Mix preparation for 5 tubes: 43,5 µl / tube

6 May 15

Results

Results from the liquid cultures: all the cultures have grown

Miniprep

Aim: plasmid purification from transformed bacteria

Protocol from MO BIO, UltraClean® Standard
Mini plasmid Prep Kit l
4 plates with Ampicillin

Dilution of the glycerol in water
Percentage in mass not in volume
Ex: 50% glycerol solution 50 g Glycerol + 50 g water (mQ)
Then filtration with vacuum pump

Glycerol preparation & Glycerol Stock

Aim: store the transformed bacteria for later use

Weight 50 g of pure Glycerol
Weight 50 g of distilled water
Mix and homogenize the solution
Filtration with vacuum pump, then close the bottle under PSM
Under PSM class II: put 1ml of liquid culture in a 1,5 ml sterile tube (pDusk, pDawn, psB1C3, VVD)
Centrifugate 1mL of liquid culture 4000 rpm, 5 min
Remove and discard the supernatant
Under PSM: resuspend with 0,5 ml of LB without antibiotics
Add 0,5 ml of glycerol 50% (25% final)
Homogenize
Transfer in an identified cryotube and store at -80°C

1 April 15

Results

Results observation for the transformed cells culture (in Petri dishes): no colonies for the positive control pUC19, other plates and negative controls are as expected.

Liquid culture

Antibiotics concentration:
Use of 3 colonies (3 tubes) from VVD, pDawn, pDusk, pSB1C3 3ml LB Broth + 3µl
Antibiotic in double click tubes (12ml) 37°C, 120rpm, O/N

31 March 15

Amplification of pDusk, pDawn, pSB1C3, VVD, pUC19 into E.coli DH5 alpha

Plate preparation

Aim: preparation of selective LB agar plate

Add 50 ml of LB agar into a Falcon tube (50ml) + 50 µl antibiotic
For 25 ml / plate (2 plates)

Agar plates preparation

Plasmid	Antibiotic	Number of plate	Remarks
pDusk
	Kanamycin	1	50 µl of transformed cells
		1	100 µl of transformed cells
		1	50 µl of competent cells (neg control)
pDawn
	Kanamycin	1	50 µl of transformed cells
		1	100 µl of transformed cells
		1	50 µl of competent cells (neg control)
pSB1C3
	Chloramphenicol	1	50 µl of transformed cells
		1	100 µl of transformed cells
		1	50 µl of competent cells (neg control)
VVD
	Amplicilin	1	50 µl of transformed cells
		1	100 µl of transformed cells
		1	50 µl of competent cells (neg control)
pUC19	Amplicilin	1	50 µl of transformed cells

Summary:
6 plates with Kanamycin
3 plates with Chloramphenicol
4 plates with Ampicillin

Antibiotics concentration:
Ampicillin: 50 µg.ml^-1 diluted 1/1000
Chloramphenicol: 25 µg.ml^-1 diluted 1/1000
Kanamycin: 50 µg.ml^-1 diluted 1/1000

Cell transformation

E.coli DH5 alpha (NEB5 alpha competent coli)
Stock: 6 tubes 0.20 ml / tube; 50 pg.µl^-1
Aliquot of 50 µl of competent E.coli / transformation

Into aliquots, added plasmids or nothing (control) as following:

Tube n°	Reagent added	Volume (µl)			Tube n°	Reagent added	Volume (µl)
1	pDusk	1			5	pSB1C3	1
2	pDawn	1			6	(Amp control)	/
3	VVD	1			7	(Kan control)	/
4	pUC19	1			8	(Cam control)	/

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min, do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 150 µl
Plate bacteria following the table about plate preparation
Incubate at 37°C, O/N

Expected results:
No colonies into the 3 control plates
Many colonies into all the other plates;
Colonies from pSB1C3 transformation should exhibit red color

30 March 15

Preparation of media

Aim: prepare media for bacteria culture

For 250mL

LB Broth	LB Agar
2.5g tryptone	2.5g tryptone
2.5g NaCl	2.5g NaCl
1.25g yeast extract	1.25g yeast extract
	5.0g agar

Qsp 250 ml distilled water
2 bottles of LB broth and 2 bottles of LB agar (+magnetic stirrer)
Auctoclave 121°C

28 March 15