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