• Protocol

• E. coli
• January
• March
• May
• June
• July
• August
• September

• L. casei
• August
• September

E. coli

January

2015/01/21

Transformation

Sakurai

Hstem

1. Added 5 μL of plac-stem-150as-stem-dt to 20 μL of thawed competent cells (DH5α) on ice.
2. Incubated on ice for 30 min.
3. Heat-shocked for 30 sec at 42℃.
4. Spread 300 μL of the culture onto plate with LBA.
5. Incubate 2ml regent with ampicillin at 37℃ for 20 hours.

2015/01/22

Colony PCR

Sakurai

Hstem

Reagent	Volume
Single Colony	-
100bp UP-EX-F 10 μM	0.8 μL
Xho-RBS-Nco 10 μM	0.8 μL
Kapa-Taq	10 μL
DW	8.4 μL
Total	20 μL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	35 cycle
Finish	68℃	60 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Sakurai

Hstem

Gel Concentration	Voltage	Time	Buffer
1%	100 A	30 min	2x TBE

Liquid Culture

Sakurai

Hstem

Reagent	Volume
Single Colony	-
LB	2 μL

Cultured for 16 hours.

2015/01/26

Colony PCR

Sakurai

Hstem

Reagent	Volume
Single Colony	-
100bp UP EX-F 10 μM	0.4 μL
Xho-RBS-Nco 10 μM	0.4 μL
Kapa-Taq	5 μL
DW	4.2 μL
Total	10 μL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	35 cycle
Finish	68℃	60 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Sakurai

Hstem

Gel Concentration	Voltage	Time	Buffer
1%	100 A	30 min	2x TBE

Colony PCR

Sakurai

Hstem

Reagent	Volume
Single Colony	-
100bp UP EX-F 10 μM	0.4 μL
Xho-RBS-Nco 10 μM	0.4 μL
Kapa-Taq	5 μL
DW	4.2 μL
Total	10 μL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	35 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	35 cycle
Finish	68℃	60 sec	Final Elongation
Store	4℃	Hold	Store

March

2015/03/10

Competent Cells

Tanaka,Sakurai

BL21 (DE3) pLysS

1. Thawed original competent cells (BL21 (DE3) pLysS) on ice.
2. Added 5 μL of original competent cells to 2 mL of LB.
3. Incubated the cells for 16 hrs at 37℃.
4. Added 5 μL, 50 μL, and 500 μL of original cells to 100 mL of LB.
5. Incubated the cells at 130 rpm for 14 hrs at 20℃, until OD₆₀₀ reach 0.5.
6. Took 50 mL of incubated cells to two differnt culture tubes and centrifuged them at 3,000 rpm for 20 min at 4℃.
7. Removed supernatant and added 75 mL of TB to each tube.
8. Brought them to a one tube and centrifuged at 3,000 rpm for 20 min at 4℃.
9. Removed supernatant and added 32 mL of TB.
10. Added 32 μL of DMSO 10 times.
11. Took 50 μL and froze with liquid nitrogen.

2015/03/11

PCR

Sakurai

BBa_R0011

Reagent	Volume
BBa_R0011	1 μL
100bpUP-EX-F 10 μM	1.5 μL
200bpDN-PS-R 10 μM	1.5 μL
KOD Plus NEO	1 μL
KOD Plus NEO 10x Buffer	5 μL
2 mM dNTPS	5 μL
25 mM MgSO4	3 μL
DW	32 μL
Total	50 μL

BBa_0030 - BBa_E1010

Reagent	Volume
BBa_0030 - BBa_E1010	1 μL
100bpUP-EX-F 10 μM	1.5 μL
200bpDN-PS-R 10 μM	1.5 μL
KOD Plus NEO	1 μL
KOD Plus NEO 10x Buffer	5 μL
2 mM dNTPS	5 μL
25 mM MgSO4	3 μL
DW	32 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	62.6℃	30 sec	Annealing	30 cycle
Cycle 3	68℃	1 min	Elongation	30 cycle
Store	4℃	Hold	Store

PCR Purification

Sakurai

BBa_R0011, BBa_0030 - BBa_E1010
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Digestion

Sakurai

BBa_R0011

Reagent	Volume
BBa_R0011	44 μL
XbaI	1 μL
Cut Smart	5 μL
Total	50 μL

BBa_0030 - BBa_E1010

Reagent	Volume
BBa_0030 - BBa_E1010	44 μL
XbaI	1 μL
Cut Smart	5 μL
Total	50 μL

Digestion

Step	Temp.	Time	Process
1	37℃	300 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

Sakurai

BBa_R0011, BBa_0030 - BBa_E1010

Gel Concentration	Voltage	Time	Buffer
1%	100 V	30 min	2x TBE

Gel Extract

Sakurai

BBa_R0011, BBa_0030 - BBa_E1010
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Electrophoresis

Sakurai

BBa_R0011, BBa_0030 - BBa_E1010

Gel Concentration	Voltage	Time	Buffer
1%	100 V	30 min	2x TBE

May

2015/05/13

Transformation

Onoda

pET15b

1. Added 1 μL of pET15b to 50 μL of thawed competent cells (DH5alpha) on ice.
2. Incubated on ice for 30 min.
3. Heat-shocked for 30 sec at 42℃.
4. Spread 50 μL of the culture onto plate with LBA.
5. Incubated the plate at 37℃ for 16 hours.

Transformation

Onoda,Sakurai

pET16b

1. Added 1 μL of pET16b to 50 μL of thawed competent cells (DH5alpha) on ice.
2. Incubated on ice for 30 min.
3. Heat-shocked for 30 sec at 42℃.
4. Spread 50 μL of the culture onto plate with LBA.
5. Incubated the plate at 37℃ for 16 hours.

Competent Cells

Onoda

rosetta

1. Thawed original competent cells (rosetta) on ice.
2. Added 5 μL of original competent cells to 2 mL of LB.
3. Incubated the cells for 16 hrs at 37℃.
4. Added 5 μL, 50 μL, and 500 μL of original cells to 100 mL of LB.
5. Incubated the cells at 130 rpm for 培養時間 hrs at 20℃, until OD₆₀₀ reach 0.5.
6. Took 50 mL of incubated cells to two differnt culture tubes and centrifuged them at 3,000 rpm for 20 min at 4℃.
7. Removed supernatant and added 75 mL of TB to each tube.
8. Brought them to a one tube and centrifuged at 3,000 rpm for 20 min at 4℃.
9. Removed supernatant and added 32 mL of TB.
10. Added 32 μL of DMSO 10 times.
11. Took 50 μL and froze with liquid nitrogen.

2015/05/27

Transformation

Mimata, Onoda, Nishimura

GFP

1. Added 1 μL of GFP to thawed competent cells (Rosetta and DH5α) on ice.
2. Incubated on ice for 30 min.
3. Heat-shocked for 30 sec at 42℃.
4. Added 200 μL of LB.
5. Spread 300 μL of the culture onto plate with LBA.
6. Incubated the plate at 37℃ for 16 hours.

Transformation

Mimata, Onoda, Ono, Nishimura

mRFP

1. Added 1 μL of mRFP to thawed competent cells (Rosetta and DH5α) on ice.
2. Incubated on ice for 30 min.
3. Heat-shocked for 30 sec at 42℃.
4. Added 200 μL of LB.
5. Spread 300 μL of the culture onto plate with LBA.
6. Incubated the plate at 37℃ for 16 hours.

2015/05/29

Mini-prep

Mimata, Onoda, Ono, Nishimura

GFP, mRFP
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

PCR

Onoda, Ono

GFP

Reagent	Volume
GFP	1 μL
67-F-primer 10 μL	1 μL
14-R-primer 10 μL	1 μL
KOD FX NEO	1 μL
KOD FX NEO 10x Buffer	5 μL
2 mM dNTPS	5 μL
25 mM MgSO4	3 μL
DW	33 μL
Total	50 μL

mRFP

Reagent	Volume
mRFP	1 μL
67-F-primer 10 μL	1 μL
14-R-primer 10 μL	1 μL
KOD FX NEO	1 μL
KOD FX NEO 10x Buffer	5 μL
2 mM dNTPS	5 μL
25 mM MgSO4	3 μL
DW	33 μL
Total	50 μL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycles
Cycle 2	68℃	60 sec	Annealing / Elongation	30 cycles
Store	4℃	Hold	Store

2015/05/30

Electrophoresis

Mimata, Onoda, Ono, Nishimura

GFP, mRFP

Gel Concentration	Voltage	Time	Buffer
2%	100 A	30 min	2x TBE

Gel Extract

Onoda, Ono, Nishimura

GFP, mRFP
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Digestion

Onoda, Ono, Nishimura

GFP

Reagent	Volume
GFP	20 μL
DW	5 μL
Spe1	1 μL
EcoR1	1 μL
Cut Smart	3 μL
Total	30 μL

mRFP

Reagent	Volume
mRFP	20 μL
DW	5 μL
Spe1	1 μL
EcoR1	1 μL
Cut Smart	3 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Onoda, Ono

pET15b

Reagent	Volume
pET15b	10 μL
DW	6 μL
Spe1	1 μL
EcoR1	1 μL
Cut Smart	2 μL
Total	20 μL

pET16b

Reagent	Volume
pET16b	10 μL
DW	6 μL
Spe1	1 μL
EcoR1	1 μL
Cut Smart	2 μL
Total	20 μL

pSB1A3

Reagent	Volume
pSB1A3	10 μL
DW	6 μL
Spe1	1 μL
EcoR1	1 μL
Cut Smart	2 μL
Total	20 μL

pSB4C5

Reagent	Volume
pSB4C5	2 μL
DW	14 μL
Spe1	1 μL
EcoR1	1 μL
Cut Smart	2 μL
Total	20 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

2015/05/31

Electrophoresis

Mimata, Onoda, Ono, Nishimura

GFP, RFP, pET15b, pET16b, pSB1A3, pSB4C5

Gel Concentration	Voltage	Time	Buffer
1%	100 V	30 min	1/2x TBE

Gel Extract

Mimata, Onoda, Ono, Nishimura

GFP, RFP, pET15b, pET16b, pSB1A3
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Electrophoresis

Mimata, Onoda, Ono, Nishimura

GFP, mRFP

Gel Concentration	Voltage	Time	Buffer
1%	100 V	30 min	1/2x TBE

PCR

Mimata, Onoda

GFP

Reagent	Volume
GFP	1 μL
67-F-primer 10 μM	1 μL
14-R-primer 10 μM	1 μL
KOD Plus NEO	1 μL
KOD Plus NEO 10x Buffer	5 μL
2 mM dNTPS	5 μL
25 mM MgSO4	3 μL
DW	33 μL
Total	50 μL

mRFP

Reagent	Volume
mRFP	1 μL
67-F-primer 10 μM	1 μL
14-R-primer 10 μM	1 μL
KOD Plus NEO	1 μL
KOD Plus NEO 10x Buffer	5 μL
2 mM dNTPS	5 μL
25 mM MgSO4	3 μL
DW	33 μL
Total	50 μL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

June

2015/06/10

Digestion

Mimata, Onoda

GFP

Reagent	Volume
GFP	16 μL
Spe1	1 μL
EcoR1	1 μL
Cut Smart	2 μL
Total	20 μL

mRFP

Reagent	Volume
mRFP	16 μL
Spe1	1 μL
EcoR1	1 μL
Cut Smart	2 μL
Total	20 μL

Digestion

Step	Temp.	Time	Process
1	37℃	600 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

2015/06/16

PCR

Mimata

thanatin fragment for TA cloning

Reagent	Volume
TA forward primer	1 μL
TA reverse primer	1 μL
Kapa Taq	25 μL
DW	23 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	180 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	35 cycle
Cycle 2	63℃	30 sec	Annealing	35 cycle
Cycle 3	72℃	10 sec	Elongation	35 cycle
	72℃	60 sec
Store	4℃	Hold	Store

2015/06/17

Electrophoresis

Mimata

thanatin fragment for TA cloning

Gel Concentration	Voltage	Time	Buffer
2%	100 A	30 min	2x TBE

→failed

Annealing Oligos and Elongation

実験者

thanatin fragment for TA cloning

Reagent	Volume
TA-forward primer 1 μM	1 μL
TA-reverse primer 1 μM	1 μL
Kapa Taq	25 μL
DW	23 μL
Total	50 μL

Annealing Oligos and Elongation

Step	Temp.	Time	Process	Cycle
Start	95℃	1 min	Initialization
Cycle1	95℃ to 23℃	45 min	Annealing	1 cycle
Cycle 2	72℃	1 min	Elongation	1 cycle
Store	4℃	Hold	Store

2015/06/19

Electrophoresis

実験者

thanatin fragment for TA cloning

Gel Concentration	Voltage	Time	Buffer
2%	100V	30min	2x TBE

Gel Extract

実験者

thanatin fragment for TA cloning
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Ligation

実験者

thanatin fragment for TA cloning / Tvector pGEM

Reagent	Volume
Tvector pGEM	1.7μL
thanatin fragment for TA cloning	0.15μL
Mighty Mix	1.85μL
T4 Ligase	0.18μL
DW	6.12μL
Total	10μL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
2	65℃	10 min	Inactivation
Store	4℃	Hold	Store

2015/06/21

Transformation

実験者

Tvector pGEM

1. Added 1 μL of thanatin fragment to 50 μL of thawed competent cells (rosseta/DH5α) on ice.
2. Incubated on ice for 30 min.
3. Heat-shocked for 30 sec at 42℃.
4. Spread 300 μL of the culture onto plate with LBA.
5. Incubated the plate at 37℃ for 19 hours.

July

2015/07/25

Transformation

Onoda

dT on pSB1C3

1. Added 1 μL of dT on pSB1C3 to 50 μL of thawed competent cells (DH5α Turbo) on ice.
2. Incubated on ice for 30 min.
3. Heat-shocked for 30 sec at 42℃.
4. Added 500 μL of LB.
5. Incubated the cells for 2 hrs at 37℃.
6. Spread 300 μL of the culture onto plate with LBCp.
7. Incubated the plate at 37℃ for 16 hours.

Transformation

Onoda

P_lac - B0034 on pSB1C3

1. Added 1 μL of P_lac - B0034 on pSB1C3 to 50 μL of thawed competent cells (DH5α Turbo) on ice.
2. Incubated on ice for 30 min.
3. Heat-shocked for 30 sec at 42℃.
4. Added 500 μL of LB.
5. Incubated the cells for 2 hrs at 37℃.
6. Spread 300 μL of the culture onto plate with LBCp.
7. Incubated the plate at 37℃ for 16 hours.

Transformation

Onoda

P_tet - B0034 on pSB1C3

1. Added 1 μL of P_tet - B0034 on pSB1C3 to 50 μL of thawed competent cells (DH5α Turbo) on ice.
2. Incubated on ice for 30 min.
3. Heat-shocked for 30 sec at 42℃.
4. Added 500 μL of LB.
5. Incubated the cells for 2 hrs at 37℃.
6. Spread 300 μL of the culture onto plate with LBCp.
7. Incubated the plate at 37℃ for 16 hours.

Transformation

Onoda

B0034 on pSB1A2

1. Added 1 μL of B0034 on pSB1A2 to 50 μL of thawed competent cells (DH5α Turbo) on ice.
2. Incubated on ice for 30 min.
3. Heat-shocked for 30 sec at 42℃.
4. Added 500 μL of LB.
5. Spread 300 μL of the culture onto plate with LBA.
6. Incubated the plate at 37℃ for 16 hours.

PCR

Onoda

dT on pSB1C3

Reagent	Volume
10% dT on pSB1C3	1 μL
100bpUP-EX-F 10 μM	1 μL
200bpDN-PS-R 10 μM	1 μL
KOD Plus NEO	1 μL
KOD Plus NEO 10x Buffer	5 μL
2 mM dNTPS	5 μL
25 mM MgSO4	3 μL
DW	33 μL
Total	50 μL

P_lac - B0034 on pSB1C3

Reagent	Volume
10% Plac - B0034 on pSB1C3	1 μL
100bpUP-EX-F 10 μM	1 μL
200bpDN-PS-R 10 μM	1 μL
KOD Plus NEO	1 μL
KOD Plus NEO 10x Buffer	5 μL
2 mM dNTPS	5 μL
25 mM MgSO4	3 μL
DW	33 μL
Total	50 μL

P_tet - B0034 on pSB1C3

Reagent	Volume
10% Ptet - B0034 on pSB1C3	1 μL
100bpUP-EX-F 10 μM	1 μL
200bpDN-PS-R 10 μM	1 μL
KOD Plus NEO	1 μL
KOD Plus NEO 10x Buffer	5 μL
2 mM dNTPS	5 μL
25 mM MgSO4	3 μL
DW	33 μL
Total	50 μL

B0034 on pSB1A2

Reagent	Volume
10% B0034 on pSB1A2	1 μL
100bpUP-EX-F 10 μM	1 μL
200bpDN-PS-R 10 μM	1 μL
KOD Plus NEO	1 μL
KOD Plus NEO 10x Buffer	5 μL
2 mM dNTPS	5 μL
25 mM MgSO4	3 μL
DW	33 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	30 cycle
Cycle 2	62.6℃	30 sec	Annealing	30 cycle
Cycle 3	68℃	60 sec	Elongation	30 cycle
Store	4℃	Hold	Store

Electrophoresis

Onoda

dT on pSB1C3, P_lac - B0034 on pSB1C3, P_tet - B0034 on pSB1C3, B0034 on pSB1A2

Gel Concentration	Voltage	Time	Buffer
2%	100 A	30 min	2x TBE

2015/07/26

Liquid Culture

Ono

P_tet - B0034 on pSB1A2

Reagent	Volume
Single Colony	-
LB	2000 μL
Ampicillin	2 μL

Cultured for 15 hours.

Mini-prep

実験者

P_lac - B0034 on pSB1C3, dT on pSB1C3, B0034 on pSB1A2
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Electrophoresis

実験者

P_tet - B0034 on pSB1C3, P_lac - B0034 on pSB1C3, B0034 on pSB1A3, dT on pSB1C3

Gel Concentration	Voltage	Time	Buffer
2%	100 A	30 min	2x TBE

Gel Extract

Ono

P_lac - B0034 on pSB1C3, P_tet - B0034 on pSB1C3, B0034 on pSB1A2, dT on pSB1C3
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

2015/07/27

Mini-prep

Ono

P_tet - B0034 on pSB1A2
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

August

September

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