Team:HokkaidoU Japan/Notebook/ecoli

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Notebook

• Protocol

• E. coli
• January
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• L. casei
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E. coli

January

2015/01/21

Transformation

Sakurai

BBa_K1524100

1. Added 5 μL of antiBBa_E1010 on BBa_K1524100 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

BBa_K1524100

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

BBa_K1524100

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

Liquid Culture

Sakurai

BBa_K1524100

Reagent	Volume
Single Colony	-
LB	2 μL

Cultured for 16 hours.

2015/01/26

Colony PCR

Sakurai

BBa_K1524100

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

BBa_K1524100

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

Colony PCR

Sakurai

BBa_K1524100

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

Colony PCR

Sakurai

BBa_K1524100

Reagent	Volume
Single Colony	-
EX-F-Universal 10 μM	0.4 μL
PS-R 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

2015/08/04

Transformation

Ito

pGEM T vector

1. Added 1 μL of pGEM T vector to 50 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. Incubated the plate at 37℃ for 16 hours.

2015/08/05

Colony PCR

Ito

pGEM T vector

Reagent	Volume
Single Colony	-
NdeⅠ - F - primer 10 μM	0.4 μL
BamHⅠ - R - primer 10 μM	0.4 μL
KAPA Taq	5.0 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℃	30 sec	Denaturation	35 cycle
Cycle 2	68℃	20 sec	Annealing / Elongation	35 cycle
Store	4℃	Hold	Store

Electrophoresis

Ito

NdeⅠ - Thanatin - BamHⅠ

Gel Concentration	Voltage	Time	Buffer
2%	100V	60 min	1/2x TBE

Gel Extract

Ito

NdeⅠ - Thanatin - BamHⅠ
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Ligation

Ito

pET vector / NdeⅠ - Thanatin - BamHⅠ

Reagent	Volume
pET vector	1 μL
NdeⅠ - Thanatin - BamHⅠ	3 μL
10 × T4 DNA Ligase buffer	5 μL
T4 Ligase	1 μL
Total	10 μL

Ligation

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

Transformation

Ito

BBa_E1010 - BBa_B0034 - BBa_E0040 - BBa_B0015 on pSB1C3

1. Added 1 μL of Thanatin on pET vector to 50 μL of thawed competent cells (Rosetta) 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 16 hours.

2015/08/10

Streaking (Single Colony Isolation)

Ito, Mimata, Mitsumoto, Onoda, Sakai

BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3, BBa_E1010 - BBa_B0034 - BBa_R0040 - BBa_B0015 on pSB1C3, BBa_E1010 - BBa_B0034 - BBa_E0400 - BBa_B0015 on pSB1C3

1. Picked the colony with an inoculating loop from the agar plate．
2. Draged the loop across on a new agar plate.
3. Re-sterilised the loop and drag it across again.

2015/08/11

Mini-prep

Ito, Mimata, Mitsumoto, Onoda, Sakai

BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
Fast protocol

Digestion

Ito, Mimata, Onoda, Sakai, Kusumi

BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3

Reagent	Volume
pSB1C3	20 μL
DW	23 μL
Bgl Ⅱ	2 μL
3.1 Buffer	5 μL
Total	50 μL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

Ito, Mimata, Onoda, Sakai, Nishimura, Kusumi

BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

2015/08/12

Gel Extract

Nishimura, Sakai

BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Electrophoresis

Ito, Sakai, Fujita

BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3 (Gel Extract Poduct)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

2015/08/13

Sequencing

Ito, Onoda, Nishimura, Fujita

BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3 (Gel Extract Product)

Reagent	Volume
pSB1C3	1 μL
T7 promoter primer / SP6 promoter primer	1.5 μL
Ready Reaction Premix	1 μL
5x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	25 cycle
Cycle 2	60℃	240 sec	-	25 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Ito, Onoda, Nishimura, Fujita, Mimata

BBa_I0500 - BBa_B0034 - BBa_K759012 (signal and β-domain) - BBa_B0015 on pSB1C3 (Sequencing PCR product)

1. Added 2 μL of NaOAc, 1.5 μL of glycogen and 50 μL of 100% ethanol.
2. Left it at -80℃ for 1 hr.
3. Centrifuged at 15,000 rpm for 15 min at 4℃.
4. Removed supernatant and added 100 μL of 70% ethanol.
5. Centrifuged at 15,000 rpm for 10 min at 4℃.
6. Removed supernatant and air-dried at room temperature with light shield.
7. Suspended with 10 μL of DW.

PCR

Ito, Onoda, Tanaka, Nishimura, Mimata

XbaⅠ - BBa_B0034 - XbaⅠ / SpeⅠ scar - Met - 10 × His tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - XbaⅠ / SpeⅠ scar - Met - 10 × His tag - Thanatin - SpeⅠ	1 μL
BamHⅠ - Thanatin forward Neo 10 μM	1 μL
BglⅡ - Asp - Thanatin reverese Neo 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	25 cycle
Cycle 2	68℃	20 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

Digestion

Mimata

NdeⅠ - Thanatin - BamHⅠ on pET vector

Reagent	Volume
NdeⅠ - Thanatin - BamHⅠ on pET vector	10 μL
NdeⅠ	1 μL
BamHⅠ	1 μL
CutSmart Buffer	5 μL
DW	33 μL
Total	50 μL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

2015/08/14

PCR

Fujita, Nishimura, Onoda

Thanatin (Mini-prep product)

Reagent	Volume
Thanatin fragment	1 μL
T7 - promoter primer 10 μM	1 μL
SP6 - promoter primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10× PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO4	3 μL
DW	33 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 66℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	94℃	10 sec	Denaturation	25 cycle
Cycle 2	50℃	10 sec	Annealing	25 cycle
Cycle 3	68℃	30 sec	Elongation	25 cycle
Store	4℃	Hold	Store

Electrophoresis

Fujita, Nishimura, Onoda, Mimata

BamHⅠ - Thanatin - BglⅡ, Thanatin fragment(PCR product)

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

Annealing of Oligonucleotides

Onoda, Nishimura, Fujita

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
TA - F - primer 10 μM	1 μL
TA - R - primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO4	3 μL
DW	34 μL
Total	50 μL

Annealing of Oligonucleotides

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

PCR

Nishimura, Onoda

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
Thanatin fragment (derived from annealing TA primers)	1 μL
BamHⅠ - Thanatin Neo 10 μM	1 μL
BglⅡ - Asp - thanatin Neo 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO4	3 μL
DW	33 μL
Total	50 μL

2 Step Cycle (Tm value ≥ 66℃)

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
Store	4℃	Hold	Store

PCR Purification

Nishimura, Onoda

Thanatin fragment from last 2 step PCR
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Electrophoresis

Nishimura, Onoda

Thanatin frament(TA-primer), Thanatin fragment(BamHⅠ/BglⅡ), Thanatin fragment(PCR Purification)

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

Transformation

Fujita, Mitsumoto

BBa_B0031 on pSB1A2, BBa_E0040 on pSB1A2

1. Added 1 μL of BBa_B0031 on pSB1A2, BBa_E0040 on pSB1A2 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. Incubated the plate at 37℃ for 12 hours.

Transformation

Fujita, Mitsumoto

BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0030

1. Added 1 μL of BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0030 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. Added 200 μL of LB.
5. Incubated the cells for 2 hrs at 37℃.
6. Spread 300 μL of the culture onto plate with LBC.
7. Incubated the plate at 37℃ for 12 hours.

PCR

Fujita, Mitsumoto

BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040

Reagent	Volume
Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040	1 μL
100UP - EX - F 10 μM	1 μL
200DN - PS - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10× PCR Buffer for KOD - Plus - Neo	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	35 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	35 cycle
Store	4℃	Hold	Store

2015/08/15

Electrophoresis

Fujita, Nishimura

BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

PCR

Fujita, Nishimura, Ono

BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040

Reagent	Volume
BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040	1 μL
100UP - EX - F 1 μM	1 μL
200DN - PS - R 1 μM	1 μL
KOD - Plus - Neo	1 μL
10× PCR Buffer for KOD - Plus - Neo	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	35 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	35 cycle
Store	4℃	Hold	Store

Electrophoresis

Fujita, Nishimura

BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

PCR

Nishimura, Ono

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
Thanatin fragment (derived from annealing TA primers)	1 μL
NdeⅠ - F - primer 10 μM	1 μL
BamHⅠ - R - primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10× PCR Buffer for KOD - Plus - Neo	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	25 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

PCR

Sakai, Ono

Thanatin fragment (PCR 2STEP product)

Reagent	Volume
Thanatin fragment (PCR 2STEP product)	1 μL
BamHⅠ - Thanatin - F - Neo 10 μM	1 μL
BglⅡ - Tanatin - R - Neo 10 μM	1 μL
KOD - Plus - Neo	1 μL
KOD Plus NEO 10 x 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	25 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

Annealing Oligos and Elongation

Onoda

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
TA - F - primer 10 μM	1 μL
TA - R - primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO4	3 μL
DW	34 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	68℃	30 sec	Elongation	1 cycle
Store	4℃	Hold	Store

Electrophoresis

Ono, Onoda

Thanatin fragment (Annealing and Elongation product), Thanatin fragment(PCR 2STEP product)

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

Gel Extract

Ono

BBa_B0031, BBa_B0030, BBa_E0040
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Mini-prep

Ono

BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E0040
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
Standard protocol

2015/08/16

PCR

Nishimura, Ono

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
Thanatin fragment (derived from annealing TA primers)	1 μL
NdeI - F - primer 10 μM	1 μL
BamHI - R - primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	25 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

Electrophoresis

Nishimura, Ono

Thanatin fragment (PCR product)

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

Colony PCR

Onoda, Ono, Nishimura

Thanatin fragment (derived from annealing TA primers) into DH5α, nothing (as a negative control)

Reagent	Volume
Single Colony	-
NdeI - F - primer 10 μM	0.4 μL
BamHI - R - primer 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	62.9℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Onoda, Ono, Nishimura

BBa_B0031 on pSB1A2 into DH5α (as positive control)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	30 cycle
Cycle 2	57.2℃	30 sec	Annealing	30 cycle
Cycle 3	72℃	30 sec	Elongation	30 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Onoda, Ono

Thanatin fragment derived from annealing TA primer (colony PCR product)

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

Electrophoresis

Ono, Mitsumoto, Fujita

BamHⅠ - Thanatin - BglⅡ, NdeⅠ - Thanatin - BamHⅠ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

PCR

Onoda

Thanatin fragment (Mini-prep product)

Reagent	Volume
Thanatin fragment (Mini-prep product)	1 μL
BamHⅠ - Thanatin - F - Neo 10 μM	1 μL
BglⅡ - Asp - Thanatin - R - Neo 10 μM	1 μL
KOD - Plus - Neo	1 μL
10× PCR Buffer for KOD - Plus - Neo	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	45 cycle
Cycle 2	65.1℃	30 sec	Annealing	45 cycle
Cycle 3	68℃	30 sec	Elongation	45 cycle
Store	4℃	Hold	Store

PCR

Onoda

Thanatin fragment (Mini-prep product)

Reagent	Volume
Thanatin fragment (Mini-prep product)	1 μL
NdeⅠ - F - primer 10 μM	1 μL
BamHⅠ - R - primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10× PCR Buffer for KOD - Plus - Neo	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	45 cycle
Cycle 2	66.5℃	30 sec	Annealing	45 cycle
Cycle 3	68℃	30 sec	Elongation	45 cycle
Store	4℃	Hold	Store

Electrophoresis

Onoda

Thanatin fragment for TA cloning and last PCR prduct

Gel Concentration	Voltage	Time	Buffer
2%	100 V	60 min	1/2 x TBE

PCR

Fujita, Mimata

Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031

Reagent	Volume
Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031	1 μL
100UP - EX - F 10 μM	1 μL
200DN - PS - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	35 cycle
Cycle 2	68℃	60 sec	Annealing / Elongation	35 cycle
Store	4℃	Hold	Store

Electrophoresis

Fujita, Mimata

Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Electrophoresis

Mitsumoto, Fujita

BamHI - Thanatin - BglⅡ, NdeI - Thanatin - BamHI

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Colony PCR

Ono, Onoda

Thanatin fragment on pGEM - T vector into DH5α

Reagent	Volume
Single Colony	-
NdeⅠ - F - primer 10 μM	0.4 μL
BamHⅠ - R - primer 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	62.9℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Ono, Onoda

Thanatin fragment on pGEM - T vector into DH5α

Reagent	Volume
Single Colony	-
T7 promoter primer 10 μM	0.4 μL
SP6 promoter primer 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	51℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Ono, Onoda

BBa_B0031 on pSB1A2 into DH5α (as positive control)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Onoda

Thanatin fragment (Colony PCR product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
TA - F - primer 10 μM	1 μL
TA - R - primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO4	3 μL
DW	34 μL
Total	50 μL

Annealing Oligos and Elongation

Step	Temp.	Time	Process	Cycle
Start	95℃	1 min	Initialization
Cycle1	95℃ to 23℃	60 sec	Annealing	45 cycle
Cycle 2	68℃	30 sec	Elongation	45 cycle
Store	4℃	Hold	Store

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
TA - F - primer 10 μM	1 μL
TA - R - primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO4	3 μL
DW	34 μL
Total	50 μL

Annealing Oligos and Elongation

3 Step Cycle (Tm value ≤ 63℃)

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

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
TA - F - primer 10 μM	1 μL
TA - R - primer 10 μM	1 μL
KOD - FX - Neo	1 μL
10× PCR Buffer for KOD - FX - Neo	25 μL
2 mM dNTPs	5 μL
25 mM MgSO4	3 μL
DW	14 μL
Total	50 μL

Annealing Oligos and Elongation

Step	Temp.	Time	Process	Cycle
Start	95℃	1 min	Initialization
Cycle1	95℃ to 23℃	60 sec	Annealing	45 cycle
Cycle 2	68℃	30 sec	Elongation	45 cycle
Store	4℃	Hold	Store

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
TA - F - primer 10 μM	1 μL
TA - R - primer 10 μM	1 μL
KOD - FX - NEO	1 μL
10× PCR Buffer for KOD - FX - Neo	25 μL
2 mM dNTPs	5 μL
25 mM MgSO4	3 μL
DW	14 μL
Total	50 μL

Annealing Oligos and Elongation

3 Step Cycle (Tm value ≤ 63℃)

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

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	1 μL
200DN - PS - R 10 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
Cycle 1	95℃ to 23℃	60 sec	Annealing	45 cycle
Cycle 2	68℃	30 sec	Elongation	45 cycle
Store	4℃	Hold	Store

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	1 μL
200DN - PS - R 10 μM	1 μL
KAPA Taq	25 μL
DW	23 μL
Total	50 μL

Annealing Oligos and Elongation

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

Electrophoresis

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

2015/08/17

Annealing Oligos and Elongation

Nishimura, Onoda

Thanatin fragment (Mini-prep product)

Reagent	Volume
TA - F - primer 10 μM	1 μL
TA - R - primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO4	3 μL
DW	34 μL
Total	50 μL

Annealing Oligos and Elongation

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	94℃	10 sec	Denaturation	10 cycle
Cycle 2	60℃	10 sec	Annealing	10 cycle
Cycle 3	68℃	3030 sec	Elongation	10 cycle
Store	4℃	Hold	Store

Annealing Oligos and Elongation

Nishimura, Onoda

Thanatin fragment (Mini-prep product)

Reagent	Volume
TA - F - primer 10 μM	1 μL
TA - R - primer 10 μM	1 μL
KAPA Taq	25 μL
DW	23 μL
Total	50 μL

(Tm value ≤ -℃)

Annealing Oligos and Elongation

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	94℃	10 sec	Denaturation	10 cycle
Cycle 2	60℃	10 sec	Annealing	10 cycle
Cycle 3	72℃	3030 sec	Elongation	10 cycle
Store	4℃	Hold	Store

PCR

Nishimura, Ono, Onoda, Mimata

NdeⅠ - Thanatin - BamHⅠ

Reagent	Volume
NdeⅠ - Thanatin - BamHⅠ	1 μL
NdeⅠ - F - primer 10 μM	1 μL
BamHⅠ - R - primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	25 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

PCR

Nishimura, Ono, Onoda, Mimata

BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - Thanatin - BglⅡ	1 μL
BamHⅠ - Asp - Thanatin - R - Neo 10 μM	1 μL
BglⅡ - D - Tanatin - R - Neo 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	25 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

PCR Purification

Ono, MImata, Nishimura

Thanatin fragment (PCR 2STEP product)
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Electrophoresis

Nishimura, Ono

Thanatin fragment (PCR 2STEP product)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

PCR

Ito, Ono, Onoda

Thanatin fragment (derived from annealing TA cloning)

Reagent	Volume
Thanatin fragment (derived from annealing TA cloning)	1 μL
NdeⅠ - F - primer 10 μM	1 μL
BamHⅠ - R - primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	45 cycle
Cycle 2	65.1℃	30 sec	Annealing	45 cycle
Cycle 3	68℃	30 sec	Elongation	45 cycle
Store	4℃	Hold	Store

PCR

Ito, Ono, Onoda

BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
Thanatin fragment (derived from annealing TA cloning)	1 μL
BamHⅠ - Asp - Thanatin - R - Neo 10 μM	1 μL
BglⅡ - D - Tanatin - R - Neo 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	45 cycle
Cycle 2	66.5℃	30 sec	Annealing	45 cycle
Cycle 3	68℃	30 sec	Elongation	45 cycle
Store	4℃	Hold	Store

Digestion

Ono, Onoda

NdeⅠ - Thanatin - BamHⅠ

Reagent	Volume
NdeⅠ - Thanatin - BamHⅠ	20 μL
NdeⅠ	1 μL
BamHⅠ	1 μL
10 × K Buffer	2 μL
DW	5 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Ono, Onoda

BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - Thanatin - BglⅡ	20 μL
BamHⅠ	1 μL
BglⅡ	1 μL
10 × K Buffer	2 μL
DW	5 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

2015/08/18

Electrophoresis

Nishimura, Ono

Thanatin fragment (PCR 2STEP product)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

PCR Purification

Ono, Mimata, Nishimura

Thanatin fragment (PCR 2STEP product)
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Annealing Oligos and Elongation

Mimata

Thanatin fragment (derived from annealing TA cloning)

Reagent	Volume
TA - F - primer 10 μM	5 μL
TA - R - primer 10 μM	5 μL
TE 0.8 M NaCl	10 μL
Total	50 μL

Annealing Oligos and Elongation

Step	Temp.	Time	Process	Cycle
Start	94℃	2 min	Initialization
Step1	95℃ to 25℃	20 min	Annealing
Store	4℃	Hold	Store

PCR

Mimata

Thanatin fragment (derived from annealing)

Reagent	Volume
Thanatin fragment (derived from annealing)	1 μL
NdeⅠ - F - primer 10 μM	1 μL
BamHⅠ - R - primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	25 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

PCR

Mimata

Thanatin fragment (derived from annealing)

Reagent	Volume
Thanatin fragment (derived from annealing)	1 μL
BamHⅠ - Asp - Thanatin - R - Neo 10 μM	1 μL
BglⅡ - D - Tanatin - R - Neo 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	25 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	25 cycle
Store	4℃	Hold	Store

PCR Purification

Ono, Mimata, Nishimura

Thanatin fragment (PCR 2STEP product)
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
Purification of PCR products

Digestion

Ono, Onoda, Nishimura

NdeⅠ - Thanatin - BamHⅠ

Reagent	Volume
NdeⅠ - Thanatin - BamHⅠ	20 μL
NdeⅠ	1 μL
BamHⅠ	1 μL
10 × K Buffer	2 μL
DW	5 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Ono, Onoda, Nishimura

BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - Thanatin - BglⅡ	20 μL
BamHⅠ	1 μL
BglⅡ	1 μL
10 × K Buffer	2 μL
DW	5 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

Ligation

Onoda, Mimata

Thanatin fragment on pGEM T - vector / Thanatin fragment (derived from annealing TA cloning)

Reagent	Volume
pGEM T - vector	1 μL
Thanatin fragment	3 μL
2 × Ligation Buffer	5 μL
T4 DNA Ligase	1 μL
Total	10 μL

Ligation

Step	Temp.	Time	Process
1	4℃	6 hour	Ligation
2	65℃	10 min	Inactivation
Store	4℃	Hold	Store

2015/08/19

PCR

Ono

Thanatin fragment (derived from annealing)

Reagent	Volume
Thanatin fragment (derived from annealing)	1 μL
NdeⅠ - F - primer 10 μM	1 μL
BamHⅠ - R - primer 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	94℃	10 sec	Denaturation	40 cycle
Cycle 2	60℃	30 sec	Annealing	40 cycle
Cycle 3	68℃	30 sec	Elongation	40 cycle
Store	4℃	Hold	Store

PCR

Ono

Thanatin fragment (derived from annealing)

Reagent	Volume
Thanatin fragment (derived from annealing)	1 μL
BamHⅠ - Thanatin - F 10 μM	1 μL
BglⅡ - Tanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	94℃	10 sec	Denaturation	40 cycle
Cycle 2	60℃	30 sec	Annealing	40 cycle
Cycle 3	68℃	30 sec	Elongation	40 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Mimata, Ono

NdeⅠ - Thanatin - BamHⅠ (PCR 3STEP product), BamHⅠ - Thanatin - BglⅡ (PCR 3STEP product)

1. Added 2 μL of NaOAc, 1 μL of glycogen and 50 μL of 100% ethanol.
2. Left it at -80℃ for 30 min.
3. Centrifuged at 15,000 rpm for 15 min at 4℃.
4. Removed supernatant and added 100 μL of 70% ethanol.
5. Centrifuged at 15,000 rpm for 10 min at 4℃.
6. Removed supernatant and air-dried at room temperature with light shield.
7. Suspended with 10 μL of TE.

Electrophoresis

Mimata

NdeⅠ - Thanatin - BamHⅠ, BamHⅠ - Thanatin - BglⅡ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

PCR

Ono

BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ	1 μL
BamHⅠ - Thanatin - F 10 μM	1 μL
BglⅡ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	40 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	40 cycle
Store	4℃	Hold	Store

PCR

Ono

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 μL
XbaⅠ - Thanatin - F 10 μM	1 μL
NdeⅠ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	40 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	40 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Ono

NdeⅠ - Thanatin - BamHⅠ (PCR 2STEP product), BamHⅠ - Thanatin - BglⅡ (PCR 2STEP product)

1. Added 2 μL of NaOAc, 1 μL of glycogen, 7 μL of DW and 50 μL of 100% ethanol.
2. Left it at room temperature for 15 min.
3. Centrifuged at 15,000 rpm for 15 min at 4℃.
4. Removed supernatant and added 100 μL of 70% ethanol.
5. Centrifuged at 15,000 rpm for 10 min at 4℃.
6. Removed supernatant and air-dried at room temperature with light shield.
7. Suspended with 10 μL of DW.

Digestion

Ono, Onoda, Mimata, Sakai

NdeⅠ - Thanatin - BamHⅠ

Reagent	Volume
NdeⅠ - Thanatin - BamHⅠ	20 μL
NdeⅠ	1 μL
BamHⅠ	1 μL
10 × K Buffer	3 μL
DW	5 μL
Total	30 μL

Digestion

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

Digestion

Ono, Onoda, Mimata, Sakai

BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - Thanatin - BglⅡ	20 μL
BamHⅠ	1 μL
BglⅡ	1 μL
10 × K Buffer	3 μL
DW	5 μL
Total	30 μL

Digestion

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

Digestion

Ono, Onoda, Mimata, Sakai

pET - 15b vector, pET - 16b vector

Reagent	Volume
pET - 15b vector, pET - 16b vector	20 μL
NdeⅠ	1 μL
BamHⅠ	1 μL
10 × K Buffer	3 μL
DW	5 μL
Total	30 μL

Digestion

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

Transformation

Onoda

Thanatin fragment on pGEM - T vector

1. Added 1 μL of Thanatin fragment on pGEM - T vector to 50 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. Incubated the plate at 37℃ for 18 hours.

2015/08/20

Electrophoresis

Ono, Nishimura, Mimata

NdeⅠ - Thanatin - BamHⅠ (digestion product), BamHⅠ - Thanatin - BglⅡ digestion product), BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (PCR 2STEP product), XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP product)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Electrophoresis

Ono, Nishimura, Mimata

pET - 15b vector (digestion product), pET - 16b vector (digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

Gel Extract

Nishimura, Ono

pET - 15b vector, pET - 16b vector
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Digestion

Ono, Nishimura, Ito

BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ(PCR 2STEP poduct)

Reagent	Volume
BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (PCR 2STEP poduct)	9 μL
BamHⅠ	5 μL
BglⅡ	5 μL
10 × K Buffer	10 μL
DW	71 μL
Total	100 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Ono, Nisimura, Ito

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP poduct)

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP poduct)	9 μL
XbaⅠ	5 μL
SpeⅠ	5 μL
10 × K Buffer	10 μL
DW	71 μL
Total	100 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Digestion

Ono, Nishimura, Ito

BBa_B0015 on pSB1C3

Reagent	Volume
BBa_B0015 on pSB1C3	20 μL
SpeⅠ	1 μL
CutSmart Buffer	3 μL
DW	6 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Ethanol Precipitation

Ito

BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (digestion product), XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (digestion product)

1. Added 9 μL of NaOAc, 1.5 μL of glycogen and 270 μL of 100% ethanol.
2. Left it at -80℃ for 1 hr.
3. Centrifuged at 15,000 rpm for 15 min at 4℃.
4. Removed supernatant and added 220 μL of 70% ethanol.
5. Centrifuged at 15,000 rpm for 10 min at 4℃.
6. Removed supernatant and air-dried at room temperature with light shield.
7. Suspended with 10 μL of TE.

Ito

Electrophoresis

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (digestion product, Ethanol Precipitation product), BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (digestion product, Ethanol Precipitation product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

2015/08/21

PCR

Ono, Nishimura, Onoda

BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ	1 μL
BamHⅠ - Thanatin - F 10 μM	1 μL
BglⅡ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	40 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	40 cycle
Store	4℃	Hold	Store

PCR

Ono, Nishimura, Onoda

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 μL
XbaⅠ - Thanatin - F 10 μM	1 μL
SpeⅠ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	40 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	40 cycle
Store	4℃	Hold	Store

Electrophoresis

Ono, Onoda, Nishimura

XbaⅠ - Thanatin - SpeⅠ, BamHⅠ- Thanatin - BglⅡ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Ethanol Precipitation

Ono, Nishimura

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ

1. Added 3 μL of NaOAc, 1 μL of glycogen and 90 μL of 100% ethanol.
2. Left it at -80℃ for 10 min.
3. Centrifuged at 15,000 rpm for 5 min at 4℃.
4. Removed supernatant and added 100 μL of 70% ethanol.
5. Centrifuged at 15,000 rpm for 5 min at 4℃.
6. Removed supernatant and air-dried at room temperature.
7. Suspended with 10 μL of TE.

Electrophoresis

Ono, Nishimura

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (after Ethanol Prescipitation) BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (after Ethanol Precipitation)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Ligation

Ono, Nishimura, Ito

BBa_K759012 on pSB1C3 / BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BBa_K759012 on pSB1C3	5 μL
BamHⅠ - Thanatin - BglⅡ	1 μL
10 × T4 DNA Ligase Buffer	7 μL
T4 DNA Ligase	1 μL
Total	14 μL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Ligation

Ono, Nishimura, Ito

BBa_B0015 on pSB1C3 / XbaⅠ - Thanatin - SpeⅠ

Reagent	Volume
BBa_B0015 on pSB1C3	5 μL
XbaⅠ - Thanatin - SpeⅠ	1 μL
10 × T4 DNA Ligase Buffer	7 μL
T4 DNA Ligase	1 μL
Total	14 μL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Transformation

Onoda

Thanatin - BBa_K759012 on pSB1C3, Thanatin - BBa_B0015 on pSB1C3

1. Added 5 μL of Thanatin - BBa_K759012 on pSB1C3, Thanatin - BBa_B0015 on pSB1C3 to 50 μL of thawed competent cells (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. Incubated the cells for 2 hrs at 37℃.
6. Spread 300 μL of the culture onto plate with LBC.
7. Incubated the plate at 37℃ for 16 hours.

Digestion

Onoda, Ono

BBa_B0033 on pSB1C3, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, XbaⅠ - BBa_K168000

Reagent	Volume
BBa_B0033 on pSB1C3, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, XbaⅠ - BBa_K168000	20 μL
XbaⅠ	1 μL
SpeⅠ	1 μL
10 × M Buffer	3 μL
DW	5 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Digestion

Onoda

BBa_E0040 on pSB1C3, BBa_R0040 on pSB1C3

Reagent	Volume
BBa_E0040 on pSB1C3, BBa_R0040 on pSB1C3	20 μL
XbaⅠ	1 μL
CutSmart Buffer	3 μL
DW	6 μL
Total	30 μL

Digestion

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

2015/08/22

Electrophoresis

Ono, Nishimura

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (digestion product)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Digestion

Ono, Onoda

BBa_R0010 - BBa_B0034 on pSB1C3

Reagent	Volume
BBa_R0010 - BBa_B0034 on pSB1C3	20 μL
SpeⅠ - HF	1 μL
10 × M Buffer	5 μL
DW	24 μL
Total	50 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
Store	4℃	Hold	Store

Electrophoresis

Ono, Onoda

BBa_R0010 - BBa_B0034 on pSB1C3 (Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	45 min	1/2 x TBE

Ethanol Precipitation

Ono

BBa_R0010 - BBa_B0034 on pSB1C3 (Digestion product)

1. Added 5 μL of NaOAc, 1.5 μL of glycogen and 150 μL of 100% ethanol.
2. Left it at -80℃ for 1 hr.
3. Centrifuged at 15,000 rpm for 5 min at 4℃.
4. Removed supernatant and added 100 μL of 70% ethanol.
5. Centrifuged at 15,000 rpm for 10 min at 4℃.
6. Removed supernatant and air-dried at room temperature with light shield.
7. Suspended with 10 μL of TE.

Colony PCR

Ono, Onoda

Thanatin - BBa_K759012 on pSB1C3, nothing (as a negative control)

Reagent	Volume
Single Colony	-
AGSP - BamHⅠ - Spidroin 10 μM	0.4 μL
BglⅡ -D - Thanatin 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	40 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Ono, Onoda

BBa_B0031 on pSB1A2 (as Positive Control)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Ono, Onoda

Thanatin - BBa_K759012 on pSB1C3 (Colony PCR product), BBa_B0031 on pSB1A2 (Colony PCR product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	45 min	1/2 x TBE

Ligation

Ono

BBa_R0010 - BBa_B0034 on pSB1C3 / XbaⅠ - Thanatin - SpeⅠ

Reagent	Volume
BBa_R0010 - BBa_B0034 on pSB1C3	5 μL
XbaⅠ - Thanatin - SpeⅠ	4 μL
Mighty Mix	10 μL
DW	1 μL
Total	20 μL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Transformation

Ono

BBa_R0010 - BBa_B0034 on pSB1C3

1. Added 1 μL of BBa_R0010 - BBa_B0034 on pSB1C3 to 50 μL of thawed competent cells (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. Incubated the cells for 2 hrs at 37℃.
6. Spread 300 μL of the culture onto plate with LBC.
7. Incubated the plate at 37℃ for 16 hours.

PCR

Onoda

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 μL
XbaI - B0034 - XS scar - F 10 μM	1 μL
SpeⅠ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

Electrophoresis

Onoda

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	50 min	1/2 x TBE

PCR

Onoda

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 μL
XbaI - B0034 - XS scar - F 10 μM	1 μL
SpeⅠ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

2015/08/23

Electrophoresis

Ono

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP product)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Gel Extract

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Colony PCR

Ito, Ono, Onoda

BBa_R0010 - BBa_B0034 - Thanatin, nothing (as a negative control)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	0.4 μL
SpeⅠ - Thanatin - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Ito, Ono, Onoda

BBa_B0030 on pSB1A2 (as a positive control)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	0.4 μL
200DN - PS - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

2015/08/24

Mini-prep

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Colony PCR

Ono, Nishimura

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3, nothing (as a negative control), BBa_B0030 on pSB1A2 (as a positive control)

Reagent	Volume
Single Colony	-
100UP - EX - F 10 μM	0.4 μL
SpeⅠ - Thanatin - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	57℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3

Reagent	Volume
Single Colony	-
AGSP - BamHⅠ - Spidroin 10 μM	0.4 μL
BBa_K759012 - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	40 sec	Annealing	40 cycle
Cycle 3	72℃	40 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Electrophoresis

Ono, Nishimura

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3, Thanatin - BBa_K759012 on pSB1C3

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Digestion

Ono, Nishimura

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3

Reagent	Volume
BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3	20 μL
SpeⅠ - HF	1 μL
CutSmart Buffer	3 μL
DW	6 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

Ono, Nishimura

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (digestion product)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Liquid Culture

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3 into DH5α

Reagent	Volume
Single Colony	-
LB	2000 μL
Chloramphenicol	2 μL

Cultured for 16 hours.

2015/08/25

Liquid Culture

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3 into DH5α, BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 into DH5α

Reagent	Volume
Single Colony	-
LB	2000 μL
Chloramphenicol	2 μL

Cultured for 16 hours.

Sequencing

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3

Reagent	Volume
Thanatin - BBa_K759012 on pSB1C3	1 μL
pbad - f2 / 200 βdomain BBa_K759012 - R	1.5 μL
BigDye Terminator	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	30 cycle
Cycle 2	60℃	240 sec	-	30 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3

1. Added 5 μL of NaOAc, 1.5 μL of glycogen and 150 μL of 100% ethanol.
2. Left it at -80℃ for 1 hr.
3. Centrifuged at 15,000 rpm for 15 min at 4℃.
4. Removed supernatant and added 220 μL of 70% ethanol.
5. Centrifuged at 15,000 rpm for 10 min at 4℃.
6. Removed supernatant and air-dried at room temperature with light shield.
7. Suspended with 10 μL of DW.

PCR

Ono, Nishimura

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 μL
XbaⅠ - BBa_B0034 - XS scar - F 10 μM	1 μL
SpeⅠ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	40 cycle
Cycle 2	55℃	10 sec	Annealing	40 cycle
Cycle 3	68℃	30 sec	Elongation	40 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Fujita, Nishimura

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

1. Added 5 μL of NaOAc, 1.5 μL of glycogen and 150 μL of 100% ethanol.
2. Left it at -80℃ for 1 hr.
3. Centrifuged at 15,000 rpm for 15 min at 4℃.
4. Removed supernatant and added 220 μL of 70% ethanol.
5. Centrifuged at 15,000 rpm for 10 min at 4℃.
6. Removed supernatant and air-dried at room temperature with light shield.
7. Suspended with 10 μL of TE.

Electrophoresis

Fujita

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

2015/08/26

PCR

Ono、Nishimura

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 μL
XbaⅠ - BBa_B0034 - XS scar - F 10 μM	1 μL
SpeⅠ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	40 cycle
Cycle 2	55℃	10 sec	Annealing	40 cycle
Cycle 3	68℃	30 sec	Elongation	40 cycle
Store	4℃	Hold	Store

PCR

Ono, Nishimura

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 μL
XbaⅠ - BBa_B0034 - XS scar - F 10 μM	1 μL
SpeⅠ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	40 cycle
Cycle 2	53℃	30 sec	Annealing	40 cycle
Cycle 3	68℃	30 sec	Elongation	40 cycle
Store	4℃	Hold	Store

PCR

Ono, Nishimura, Fujita

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 μL
XbaⅠ - BBa_B0034 - XS scar - F 10 μM	1 μL
SpeⅠ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

Electrophoresis

Ono, Nishimura, Fujita

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP, 3STEP products)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Ethanol Precipitation

Fujita, Nishimura, Ono

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP product)

1. Added 4 μL of NaOAc, 1.5 μL of glycogen and 120 μL of 100% ethanol.
2. Left it at -80℃ for 1 hr.
3. Centrifuged at 15,000 rpm for 5 min at 4℃.
4. Removed supernatant and added 100 μL of 70% ethanol.
5. Centrifuged at 15,000 rpm for 10 min at 4℃.
6. Removed supernatant and air-dried at room temperature with light shield.
7. Suspended with 10 μL of TE.

Mini-prep

Nishimura, Ono

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3
FastGene^TM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Sequencing

Fujita, Nishimura, Ono

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (Mini-prep product)

Reagent	Volume
BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (Mini-prep producrt)	1 μL
100UP - EX - F / 200DN - PS - R	1.5 μL
Ready Reaction Premix	1 μL
5 x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	30 cycle
Cycle 2	60℃	240 sec	-	30 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Fujita, Nishimura, Ono

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (Sequencing PCR product)

1. Added 5 μL of NaOAc, 1.5 μL of glycogen and 150 μL of 100% ethanol.
2. Left it at -80℃ for 1 hr.
3. Centrifuged at 15,000 rpm for 15 min at 4℃.
4. Removed supernatant and added 220 μL of 70% ethanol.
5. Centrifuged at 15,000 rpm for 10 min at 4℃.
6. Removed supernatant and air-dried at room temperature with light shield.
7. Suspended with 10 μL of TE.

2015/08/27

Ligation

Ono, Ito

BBa_K759012 on pSB1C3 (dephosphorylated) / BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BBa_K759012 on pSB1C3	5 μL
BamHⅠ - Thanatin - BglⅡ	4 μL
Mighty Mix	10 μL
DW	1 μL
Total	20 μL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Ligation

Ono, Ito

BBa_K759012 on pSB1C3 (not phosphorylated)

Reagent	Volume
BBa_K759012 on pSB1C3	2 μL
Mighty Mix	2 μL
DW	6 μL
Total	10 μL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Ligation

Ono, Ito

BBa_K759012 on pSB1C3 (phosphorylated)

Reagent	Volume
BBa_K759012 on pSB1C3	2 μL
Mighty Mix	2 μL
DW	6 μL
Total	10 μL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Transformation

Ono, Ito

Thanatin - BBa_K759012 on pSB1C3, self ligated BBa_K759012 on pSB1C3, linearized BBa_K759012 on pSB1C3, self ligated BBa_K759012 on pSB1C3 (phosphorylated), linearized BBa_K759012 on pSB1C3 (phosphorylated)

1. Added 1 μL of Thanatin - BBa_K759012 on pSB1C3, self ligated BBa_K759012 on pSB1C3, linearized BBa_K759012 on pSB1C3, self ligated BBa_K759012 on pSB1C3 (phosphorylated), linearized BBa_K759012 on pSB1C3 (phosphorylated) or linearized BBa_K759012 on pSB1C3 (phosphorylated) to 50 μL of thawed competent cells (DH5a) on ice.
2. Incubated on ice for 30 min.
3. Heat-shocked for 30 sec at 42℃.
4. Added 200 μL of LB.
5. Incubated the cells for 2 hrs at 37℃.
6. Spread 300 μL of the culture onto plate with LBC.
7. Incubated the plate at 37℃ for 12 hours.

Colony PCR

Ito, Ono

Thanatin - BBa_K759012 on pSB1C3

Reagent	Volume
Single Colony	-
Agsp - BamHⅠ - Spidroin 10 μM	0.4 μL
BBa_K759012 - bunit - R / BglⅡ - D - Thanatin - R 10 μM	0.4 μL
KAPA Taq	5 μL
DW	4.2 μL
Total	10 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	40 cycle
Cycle 2	65℃	30 sec	Annealing	40 cycle
Cycle 3	72℃	60 sec	Elongation	40 cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

2015/08/28

Electrophoresis

Fujita, Ono

BamHⅠ - Thanatin - BglⅡ, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Ethanol Precipitation

Fujita

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

1. Added 5 μL of NaOAc, 1.5 μL of glycogen and 150 μL of 100% ethanol.
2. Left it at -80℃ for 1 hr.
3. Centrifuged at 15,000 rpm for 15 min at 4℃.
4. Removed supernatant and added 220 μL of 70% ethanol.
5. Centrifuged at 15,000 rpm for 10 min at 4℃.
6. Removed supernatant and air-dried at room temperature with light shield.
7. Suspended with 10 μL of TE.

Electrophoresis

Fujita, Ono

BamHⅠ - Thanatin - BglⅡ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Gel Extract

Fujita

BamHⅠ - Thanatin - BglⅡ
FastGene^TM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

PCR

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - Thanatin - SpeⅠ	1 μL
XbaⅠ - Thanatin - F 10 μM	1 μL
SpeⅠ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 μL
XbaⅠ - Thanatin - F 10 μM	1 μL
SpeⅠ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	67℃	30 sec	Annealing	30 cycle
Cycle 3	68℃	30 sec	Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Fujita, Ono

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 μL
XbaⅠ - Thanatin - F 10 μM	1 μL
SpeⅠ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Fujita, Ono

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 μL
XbaⅠ - Thanatin - F 10 μM	1 μL
SpeⅠ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	67℃	30 sec	Annealing	30 cycle
Cycle 3	68℃	30 sec	Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Fujita, Ono

BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - Thanatin - BglⅡ	1 μL
BamHⅠ - Thanatin - F 10 μM	1 μL
BglⅡ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	5 μL
2 mM dNTPs	5 μL
25 mM MgSO4	3 μL
DW	35 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℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Fujita, Ono

BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - Thanatin - BglⅡ	1 μL
BamHⅠ - Thanatin - F 10 μM	1 μL
BglⅡ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	67℃	30 sec	Annealing	30 cycle
Cycle 3	68℃	30 sec	Elongation	30 cycle
Store	4℃	Hold	Store

Electrophoresis

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, BamHⅠ - Thanatin - BglⅡ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Digestion

Ono, Fujita

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	20 μL
SpeⅠ	1 μL
XbaⅠ	1 μL
10 × M Buffer	3 μL
0.1%BSA	3 μL
DW	2 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
Store	4℃	Hold	Store

Digestion

Ono, Fujita

BamHⅠ - Thanatin - BglⅡ

Reagent	Volume
BamHⅠ - Thanatin - BglⅡ	20 μL
BamHⅠ	1 μL
BglⅡ	1 μL
10 × K Buffer	3 μL
DW	5 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
Store	4℃	Hold	Store

2015/08/29

Electrophoresis

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Gel Extract

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

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

Ethanol Precipitation

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

1. Added 20 μL of NaOAc, 1.5 μL of glycogen and 600 μL of 100% ethanol.
2. Left it at -80℃ for 1 hr.
3. Centrifuged at 15,000 rpm for 15 min at 4℃.
4. Removed supernatant and added 100 μL of 70% ethanol.
5. Centrifuged at 15,000 rpm for 10 min at 4℃.
6. Removed supernatant and air-dried at room temperature with light shield.
7. Suspended with 10 μL of TE.

Electrophoresis

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

PCR

Ono

XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ	1 μL
XbaⅠ - Thanatin - F 10 μM	1 μL
SpeⅠ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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	40 cycle
Cycle 2	68℃	30 sec	Annealing / Elongation	40 cycle
Store	4℃	Hold	Store

Ethanol Precipitation

Ono

BamHⅠ - Thanatin - BglⅡ (digestion product), XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (digestion product)

1. Added 3 μL of NaOAc, 1.5 μL of glycogen and 90 μL of 100% ethanol.
2. Left it at -80℃ for 10 min.
3. Centrifuged at 15,000 rpm for 5 min at 4℃.
4. Removed supernatant and added 100 μL of 70% ethanol.
5. Centrifuged at 15,000 rpm for 5 min at 4℃.
6. Removed supernatant and air-dried at room temperature with light shield.
7. Suspended with 10 μL of TE.

Electrophoresis

Fujita, Mimata

BBa_B0033, Thanatin - BBa_K759012, BBa_R0010 - Thanatin, BBa_R0040, BBa_E0040

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

Electrophoresis

Fujita, Mimata

BamHⅠ - Thanatin - BglⅡ, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, XbaⅠ - Thanatin - SpeⅠ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Digestion

Fujita

BBa_R0010 - BBa_B0034 on pSB1C3

Reagent	Volume
BBa_R0010 - BBa_B0034 on pSB1C3	20 μL
SpeⅠ - HF	1 μL
CutSmart Buffer	3 μL
DW	6 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

2015/08/30

Electrophoresis

Mimata

BBa_R0010 - BBa_B0034 on pSB1C3, Thanatin - BBa_K759012 on pSB1C3

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

Digestion

Mimata, Toyooka

XbaⅠ - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - Thanatin - SpeⅠ	20 μL
SpeⅠ	1 μL
XbaⅠ	1 μL
10 × M Buffer	3 μL
DW	5 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Mimata, Toyooka

BBa_R0040 on pSB1A2, BBa_E0040 on pSB1A2

Reagent	Volume
BBa_R0040 on pSB1A2, BBa_E0040 on pSB1A2	20 μL
SpeⅠ	1 μL
CutSmart Buffer	3 μL
DW	6 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

Digestion

Mimata, Toyooka

BBa_B0030 on pSB1C3

Reagent	Volume
BBa_B0030 on pSB1C3	20 μL
SpeⅠ	1 μL
XbaⅠ	1 μL
10 × M Buffer	3 μL
DW	5 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	70℃	15 min	Inactivation
Store	4℃	Hold	Store

2015/08/31

PCR

Nishimura, Ono, Toyooka, Fujita

XbaⅠ - Thanatin - SpeⅠ

Reagent	Volume
XbaⅠ - Thanatin - SpeⅠ	1 μL
XbaⅠ - Thanatin - F 10 μM	1 μL
SpeⅠ - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

PCR

Nishimura, Ono, Toyooka, Fujita

BamHⅠ- Thanatin - BglⅡ

Reagent	Volume
BamHⅠ- Thanatin - BglⅡ	1 μL
BamHⅠ- Thanatin - F 10 μM	1 μL
BglⅡ - D - Thanatin - R 10 μM	1 μL
KOD - Plus - Neo	1 μL
10 x PCR Buffer for KOD - Plus - Neo	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℃	30 sec	Annealing / Elongation	30 cycle
Store	4℃	Hold	Store

Electrophoresis

Ono, Nishimura, Toyooka, Fujita

BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Ethanol Precipitation

Ono, Nishimura, Toyooka, Fujita

BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ

1. Added 5 μL of NaOAc, 1.5 μL of glycogen and 150 μL of 100% ethanol.
2. Left it at -80℃ for 1 hr.
3. Centrifuged at 15,000 rpm for 15 min at 4℃.
4. Removed supernatant and added 100 μL of 70% ethanol.
5. Centrifuged at 15,000 rpm for 10 min at 4℃.
6. Removed supernatant and air-dried at room temperature with light shield.
7. Suspended with 10 μL of TE.

Digestion

Ono, Fujita, Toyooka, Nishimura

Thanatin fragment (Ethanol Precipitation product)

Reagent	Volume
Thanatin fragment (Ethanol Precipitation product)	20 μL
SpeⅠ	2 μL
XbaⅠ	1 μL
CutSmart Buffer	3 μL
DW	4 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
Store	4℃	Hold	Store

Digestion

Ono, Fujita, Toyooka, Nishimura

Thanatin fragment (Ethanol Precipitation product)

Reagent	Volume
Thanatin fragment (Ethanol Precipitation product)	20 μL
BamHⅠ	2 μL
BglⅡ	6 μL
10 × K Buffer	10 μL
DW	60 μL
Total	100 μL

Digestion

Step	Temp.	Time	Process
1	37℃	60 min	Digestion
Store	4℃	Hold	Store

Ethanol Precipitation

Ono, Nishimura, Toyooka, Fujita

BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ (Digestion product)

1. Added 5 μL of NaOAc, 1.5 μL of glycogen and 280 μL of 100% ethanol.
2. Left it at -80℃ for 1 hr.
3. Centrifuged at 15,000 rpm for 15 min at 4℃.
4. Removed supernatant and added 100 μL of 70% ethanol.
5. Centrifuged at 15,000 rpm for 10 min at 4℃.
6. Removed supernatant and air-dried at room temperature with light shield.
7. Suspended with 10 μL of TE.

Electrophoresis

Ono, Nishimura, Toyooka, Fujita, Mimata

BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ (Ethanol Presipitation product)

Gel Concentration	Voltage	Time	Buffer
2%	50 V	60 min	1/2 x TBE

Ligation

Ono

BBa_K759012 on pSB1C3 / BamHⅠ- Thanatin - BglⅡ

Reagent	Volume
BBa_K759012 on pSB1C3	95 μL
BamHⅠ- Thanatin - BglⅡ	0.5 μL
Mighty Mix	10 μL
Total	20 μL

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
Store	4℃	Hold	Store

Electrophoresis

Ono, Mimata

BBa_K759012 on pSB1C3, BBa_R0010 on pSB1C3

Gel Concentration	Voltage	Time	Buffer
1%	100 V	60 min	1/2 x TBE

Digestion

Onoda,

BBa_E1010 on pSB1C3

Reagent	Volume
BBa_E1010 on pSB1C3	10 μL
EcoRⅠ	1 μL
XbaⅠ	1 μL
10 × M Buffer	2 μL
DW	6 μL
Total	20 μL

Digestion

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

Digestion

Onoda

BBa_E1010 on pSB1C3

Reagent	Volume
BBa_E1010 on pSB1C3	10 μL
XbaⅠ	1 μL
CutSmart Buffer	2 μL
DW	7 μL
Total	20 μL

Digestion

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

Electrophoresis

Toyooka

BBa_E1010 on pSB1C3 (Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Gel Extract

Toyooka

BBa_E1010 on pSB1C3 (Digestion product)
FastGene^TM Gel Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

Digestion

Ito, Sakai

HLA family

Reagent	Volume
HLA, HLZ, BLA, BLZ	20 μL
XbaⅠ	1 μL
SpeⅠ	1 μL
10 × M Buffer	3 μL
DW	5 μL
Total	30 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Digestion

Ito, Sakai

HLA, HLZ, BLA, BLZ

Reagent	Volume
HLA, HLZ, BLA, BLZ	10 μL
XbaⅠ	1 μL
SpeⅠ	1 μL
10 x M buffer	2 μL
DW	6 μL
Total	20 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	80℃	20 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

Ito, Sakai

HLA family XbaⅠ & SpeⅠ (Digestion product)

Gel Concentration	Voltage	Time	Buffer
1%	100 V	40 min	1/2 x TBE

Gel Extract

Ito, Sakai

HLA family XbaⅠ & SpeⅠ (Digestion product)
FastGene^TM Gel Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

September

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