Team:HokkaidoU Japan/Notebook/ecoli
E. coli
January
2015/01/21
Transformation
Sakurai
BBa_K1524100
- Added 5 μL of antiBBa_E1010 on BBa_K1524100 to 20 μL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Spread 300 μL of the culture onto plate with LBA.
- Incubate 2ml regent with ampicillin at 37℃ for 20 hours.
2015/01/22
Colony PCR
Sakurai
BBa_K1524100
Reagent | Volume |
---|---|
Single Colony | - |
100UP - EX - F 10 μM | 0.8 μL |
XhoⅠ - RBS - NcoⅠ 10 μM | 0.8 μL |
TAKA 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 V | 30 min | 1/2 x 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 | - |
100UP - EX -F 10 μM | 0.4 μL |
XhoⅠ - RBS - NcoⅠ 10 μM | 0.4 μL |
TAKA 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 V | 30 min | 1/2 x TBE |
Colony PCR
Sakurai
BBa_K1524100
Reagent | Volume |
---|---|
Single Colony | - |
100UP - EX - F 10 μM | 0.4 μL |
XhoⅠ - RBS - NcoⅠ 10 μM | 0.4 μL |
TAKA 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 |
TAKA 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
- Thawed original competent cells (BL21 (DE3) pLysS) on ice.
- Added 5 μL of original competent cells to 2 mL of LB.
- Incubated the cells for 16 hrs at 37℃.
- Added 5 μL, 50 μL, and 500 μL of original cells to 100 mL of LB.
- Incubated the cells at 130 rpm for 14 hrs at 20℃, until OD600 reach 0.5.
- Took 50 mL of incubated cells to two differnt culture tubes and centrifuged them at 3,000 rpm for 20 min at 4℃.
- Removed supernatant and added 75 mL of TB to each tube.
- Brought them to a one tube and centrifuged at 3,000 rpm for 20 min at 4℃.
- Removed supernatant and added 32 mL of TB.
- Added 32 μL of DMSO 10 times.
- Took 50 μL and froze with liquid nitrogen.
2015/03/11
PCR
Sakurai
BBa_R0011
Reagent | Volume |
---|---|
BBa_R0011 | 1 μL |
100UP - EX - F 10 μM | 1.5 μL |
200DN - PS - R 10 μM | 1.5 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 | 32 μL |
Total | 50 μL |
BBa_0030 - BBa_E1010
Reagent | Volume |
---|---|
BBa_0030 - BBa_E1010 | 1 μL |
100UP - EX - F 10 μM | 1.5 μL |
200DN - PS - R 10 μM | 1.5 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 | 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
FastGeneTM 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 |
CutSmart Buffer | 5 μL |
Total | 50 μL |
BBa_0030 - BBa_E1010
Reagent | Volume |
---|---|
BBa_0030 - BBa_E1010 | 44 μL |
XbaI | 1 μL |
CutSmart Buffer | 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 | 1/2 x TBE |
Gel Extract
Sakurai
BBa_R0011, BBa_0030 - BBa_E1010
FastGeneTM 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 | 1/2 x TBE |
May
2015/05/13
Transformation
Onoda
pET15b
- Added 1 μL of pET15b to 50 μL of thawed competent cells (DH5alpha) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Spread 50 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 16 hours.
Transformation
Onoda, Sakurai
pET16b
- Added 1 μL of pET16b to 50 μL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Spread 50 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 16 hours.
Competent Cells
Onoda
Rosetta
- Thawed original competent cells (Rosetta) on ice.
- Added 5 μL of original competent cells to 2 mL of LB.
- Incubated the cells for 16 hrs at 37℃.
- Added 5 μL, 50 μL, and 500 μL of original cells to 100 mL of LB.
- Incubated the cells at 130 rpm for 24 hrs at 20℃, until OD600 reach 0.5.
- Took 50 mL of incubated cells to two differnt culture tubes and centrifuged them at 3,000 rpm for 20 min at 4℃.
- Removed supernatant and added 75 mL of TB to each tube.
- Brought them to a one tube and centrifuged at 3,000 rpm for 20 min at 4℃.
- Removed supernatant and added 32 mL of TB.
- Added 32 μL of DMSO 10 times.
- Took 50 μL and froze with liquid nitrogen.
2015/05/27
Transformation
Mimata, Onoda, Nishimura
BBa_E0040
- Added 1 μL of BBa_E0040 to thawed competent cells (Rosetta and DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Spread 300 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 16 hours.
Transformation
Mimata, Onoda, Ono, Nishimura
mBBa_R0040
- Added 1 μL of mBBa_R0040 to thawed competent cells (Rosetta and DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Spread 300 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 16 hours.
2015/05/29
Mini-prep
Mimata, Onoda, Ono, Nishimura
BBa_E0040, mBBa_R0040
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol
PCR
Onoda, Ono
BBa_E0040
Reagent | Volume |
---|---|
BBa_E0040 | 1 μL |
EX - F - Universal 10 μM | 1 μL |
200DN - PS - R 10 μM | 1 μL |
KOD - FX - Neo | 1 μL |
10x PCR Buffer for KOD - FX - Neo | 5 μL |
2 mM dNTPs | 5 μL |
25 mM MgSO4 | 3 μL |
DW | 33 μL |
Total | 50 μL |
mBBa_R0040
Reagent | Volume |
---|---|
mBBa_R0040 | 1 μL |
EX - F - Universal 10 μM | 1 μL |
200DN - PS - R 10 μM | 1 μL |
KOD - FX - Neo | 1 μL |
10x PCR Buffer for KOD - FX - 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 cycles |
Cycle 2 | 68℃ | 60 sec | Annealing / Elongation | 30 cycles |
Store | 4℃ | Hold | Store |
2015/05/30
Electrophoresis
Mimata, Onoda, Ono, Nishimura
BBa_E0040, mBBa_R0040
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
2% | 100 V | 30 min | 1/2 x TBE |
Gel Extract
Onoda, Ono, Nishimura
BBa_E0040, mBBa_R0040
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
Digestion
Onoda, Ono, Nishimura
BBa_E0040
Reagent | Volume |
---|---|
BBa_E0040 | 20 μL |
DW | 5 μL |
SpeⅠ | 1 μL |
EcoRⅠ | 1 μL |
CutSmart Buffer | 3 μL |
Total | 30 μL |
mBBa_R0040
Reagent | Volume |
---|---|
mBBa_R0040 | 20 μL |
DW | 5 μL |
SpeⅠ | 1 μL |
EcoRⅠ | 1 μL |
CutSmart Buffer | 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 |
SpeⅠ | 1 μL |
EcoRⅠ | 1 μL |
CutSmart Buffer | 2 μL |
Total | 20 μL |
pET16b
Reagent | Volume |
---|---|
pET16b | 10 μL |
DW | 6 μL |
SpeⅠ | 1 μL |
EcoRⅠ | 1 μL |
CutSmart Buffer | 2 μL |
Total | 20 μL |
pSB1A3
Reagent | Volume |
---|---|
pSB1A3 | 10 μL |
DW | 6 μL |
SpeⅠ | 1 μL |
EcoRⅠ | 1 μL |
CutSmart Buffer | 2 μL |
Total | 20 μL |
pSB4C5
Reagent | Volume |
---|---|
pSB4C5 | 2 μL |
DW | 14 μL |
SpeⅠ | 1 μL |
EcoRⅠ | 1 μL |
CutSmart Buffer | 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
BBa_E0040, BBa_R0040, pET15b, pET16b, pSB1A3, pSB4C5
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
1% | 100 V | 30 min | 1/2 x TBE |
Gel Extract
Mimata, Onoda, Ono, Nishimura
BBa_E0040, BBa_R0040, pET15b, pET16b, pSB1A3
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
Electrophoresis
Mimata, Onoda, Ono, Nishimura
BBa_E0040, mBBa_R0040
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
1% | 100 V | 30 min | 1/2 x TBE |
PCR
Mimata, Onoda
BBa_E0040
Reagent | Volume |
---|---|
BBa_E0040 | 1 μL |
EX - F - Universal 10 μM | 1 μL |
200DN - 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 |
mBBa_R0040
Reagent | Volume |
---|---|
mBBa_R0040 | 1 μL |
EX - F - Universal 10 μM | 1 μL |
200DN - 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 |
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
BBa_E0040
Reagent | Volume |
---|---|
BBa_E0040 | 16 μL |
SpeⅠ | 1 μL |
EcoRⅠ | 1 μL |
CutSmart Buffer | 2 μL |
Total | 20 μL |
mBBa_R0040
Reagent | Volume |
---|---|
mBBa_R0040 | 16 μL |
SpeⅠ | 1 μL |
EcoRⅠ | 1 μL |
CutSmart Buffer | 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 - F - primer | 1 μL |
TA - R - 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 V | 30 min | 1/2 x TBE |
Annealing Oligos and Elongation
Ito
thanatin fragment for TA cloning
Reagent | Volume |
---|---|
TA - F - primer 1 μM | 1 μL |
TA - R - 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
Ito
thanatin fragment for TA cloning
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
2% | 100 V | 30min | 1/2 x TBE |
Gel Extract
Ito
thanatin fragment for TA cloning
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
Ligation
Ito
pGEM - T vector / Thanatin fragment for TA cloning
Reagent | Volume |
---|---|
pGEM - T vector | 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
Ito
pGEM - T vector
- Added 1 μL of Thanatin fragment to 50 μL of thawed competent cells (Rosseta/DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Spread 300 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 19 hours.
July
2015/07/25
Transformation
Onoda
BBa_B0015 on pSB1C3
- Added 1 μL of BBa_B0015 on pSB1C3 to 50 μL of thawed competent cells (DH5α Turbo) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 500 μL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 μL of the culture onto plate with LBC.
- Incubated the plate at 37℃ for 16 hours.
Transformation
Onoda
BBa_R0010 - BBa_B0034 on pSB1C3
- Added 1 μL of BBa_R0010 - BBa_B0034 on pSB1C3 to 50 μL of thawed competent cells (DH5α Turbo) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 500 μL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 μL of the culture onto plate with LBC.
- Incubated the plate at 37℃ for 16 hours.
Transformation
Onoda
BBa_I0500 - BBa_B0034 on pSB1C3
- Added 1 μL of BBa_I0500 - BBa_B0034 on pSB1C3 to 50 μL of thawed competent cells (DH5α Turbo) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 500 μL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 μL of the culture onto plate with LBC.
- Incubated the plate at 37℃ for 16 hours.
Transformation
Onoda
BBa_B0034 on pSB1A2
- Added 1 μL of BBa_B0034 on pSB1A2 to 50 μL of thawed competent cells (DH5α Turbo) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 500 μL of LB.
- Spread 300 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 16 hours.
PCR
Onoda
BBa_B0015 on pSB1C3
Reagent | Volume |
---|---|
BBa_B0015 on pSB1C3 | 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 |
BBa_R0010 - BBa_B0034 on pSB1C3
Reagent | Volume |
---|---|
BBa_R0010 - BBa_B0034 on pSB1C3 | 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 |
BBa_I0500 - BBa_B0034 on pSB1C3
Reagent | Volume |
---|---|
BBa_I0500 - BBa_B0034 on pSB1C3 | 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 |
BBa_B0034 on pSB1A2
Reagent | Volume |
---|---|
BBa_B0034 on pSB1A2 | 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 |
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
BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 on pSB1C3, BBa_I0500 - BBa_B0034 on pSB1C3, BBa_B0034 on pSB1A2
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
2% | 100 V | 30 min | 1/2 x TBE |
2015/07/26
Liquid Culture
Ono
BBa_I0500 - BBa_B0034 on pSB1A2
Reagent | Volume |
---|---|
Single Colony | - |
LB | 2000 μL |
Ampicillin | 2 μL |
Cultured for 15 hours.
Mini-prep
Ito
BBa_R0010 - BBa_B0034 on pSB1C3, BBa_B0015 on pSB1C3, BBa_B0034 on pSB1A2
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol
Electrophoresis
Ito
BBa_I0500 - BBa_B0034 on pSB1C3, BBa_R0010 - BBa_B0034 on pSB1C3, BBa_B0034 on pSB1A3, BBa_B0015 on pSB1C3
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
2% | 100 V | 30 min | 1/2 x TBE |
Gel Extract
Ono
BBa_R0010 - BBa_B0034 on pSB1C3, BBa_I0500 - BBa_B0034 on pSB1C3, BBa_B0034 on pSB1A2, BBa_B0015 on pSB1C3
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
2015/07/27
Mini-prep
Ono
BBa_I0500 - BBa_B0034 on pSB1A2
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol
August
2015/08/04
Transformation
Ito
pGEM T vector
- Added 1 μL of pGEM T vector to 50 μL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Spread 300 μL of the culture onto plate with LBA.
- 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
FastGeneTM 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
- Added 1 μL of Thanatin on pET vector to 50 μL of thawed competent cells (Rosetta) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Spread 300 μL of the culture onto plate with LBA.
- 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
- Picked the colony with an inoculating loop from the agar plate.
- Draged the loop across on a new agar plate.
- 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
FastGeneTM 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
FastGeneTM 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)
- Added 2 μL of NaOAc, 1.5 μL of glycogen and 50 μL of 100% ethanol.
- Left it at -80℃ for 1 hr.
- Centrifuged at 15,000 rpm for 15 min at 4℃.
- Removed supernatant and added 100 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 10 min at 4℃.
- Removed supernatant and air-dried at room temperature with light shield.
- 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
FastGeneTM 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
- Added 1 μL of BBa_B0031 on pSB1A2, BBa_E0040 on pSB1A2 to 20 μL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Spread 300 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 12 hours.
Transformation
Fujita, Mitsumoto
BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0030
- 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.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 μL of the culture onto plate with LBC.
- 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
FastGeneTM 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
FastGeneTM 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
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
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 ≤ -℃)
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)
FastGeneTM 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)
FastGeneTM 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)
FastGeneTM 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)
- Added 2 μL of NaOAc, 1 μL of glycogen and 50 μL of 100% ethanol.
- Left it at -80℃ for 30 min.
- Centrifuged at 15,000 rpm for 15 min at 4℃.
- Removed supernatant and added 100 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 10 min at 4℃.
- Removed supernatant and air-dried at room temperature with light shield.
- 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)
- Added 2 μL of NaOAc, 1 μL of glycogen, 7 μL of DW and 50 μL of 100% ethanol.
- Left it at room temperature for 15 min.
- Centrifuged at 15,000 rpm for 15 min at 4℃.
- Removed supernatant and added 100 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 10 min at 4℃.
- Removed supernatant and air-dried at room temperature with light shield.
- 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
- Added 1 μL of Thanatin fragment on pGEM - T vector to 50 μL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Spread 300 μL of the culture onto plate with LBA.
- 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
FastGeneTM 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)
- Added 9 μL of NaOAc, 1.5 μL of glycogen and 270 μL of 100% ethanol.
- Left it at -80℃ for 1 hr.
- Centrifuged at 15,000 rpm for 15 min at 4℃.
- Removed supernatant and added 220 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 10 min at 4℃.
- Removed supernatant and air-dried at room temperature with light shield.
- 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
- Added 3 μL of NaOAc, 1 μL of glycogen and 90 μL of 100% ethanol.
- Left it at -80℃ for 10 min.
- Centrifuged at 15,000 rpm for 5 min at 4℃.
- Removed supernatant and added 100 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 5 min at 4℃.
- Removed supernatant and air-dried at room temperature.
- 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
- Added 5 μL of Thanatin - BBa_K759012 on pSB1C3, Thanatin - BBa_B0015 on pSB1C3 to 50 μL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 μL of the culture onto plate with LBC.
- 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)
- Added 5 μL of NaOAc, 1.5 μL of glycogen and 150 μL of 100% ethanol.
- Left it at -80℃ for 1 hr.
- Centrifuged at 15,000 rpm for 5 min at 4℃.
- Removed supernatant and added 100 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 10 min at 4℃.
- Removed supernatant and air-dried at room temperature with light shield.
- 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
- Added 1 μL of BBa_R0010 - BBa_B0034 on pSB1C3 to 50 μL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 μL of the culture onto plate with LBC.
- 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
FastGeneTM 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
FastGeneTM 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
- Added 5 μL of NaOAc, 1.5 μL of glycogen and 150 μL of 100% ethanol.
- Left it at -80℃ for 1 hr.
- Centrifuged at 15,000 rpm for 15 min at 4℃.
- Removed supernatant and added 220 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 10 min at 4℃.
- Removed supernatant and air-dried at room temperature with light shield.
- 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
- Added 5 μL of NaOAc, 1.5 μL of glycogen and 150 μL of 100% ethanol.
- Left it at -80℃ for 1 hr.
- Centrifuged at 15,000 rpm for 15 min at 4℃.
- Removed supernatant and added 220 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 10 min at 4℃.
- Removed supernatant and air-dried at room temperature with light shield.
- 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)
- Added 4 μL of NaOAc, 1.5 μL of glycogen and 120 μL of 100% ethanol.
- Left it at -80℃ for 1 hr.
- Centrifuged at 15,000 rpm for 5 min at 4℃.
- Removed supernatant and added 100 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 10 min at 4℃.
- Removed supernatant and air-dried at room temperature with light shield.
- Suspended with 10 μL of TE.
Mini-prep
Nishimura, Ono
BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3
FastGeneTM 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)
- Added 5 μL of NaOAc, 1.5 μL of glycogen and 150 μL of 100% ethanol.
- Left it at -80℃ for 1 hr.
- Centrifuged at 15,000 rpm for 15 min at 4℃.
- Removed supernatant and added 220 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 10 min at 4℃.
- Removed supernatant and air-dried at room temperature with light shield.
- 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)
- 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.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 μL of the culture onto plate with LBC.
- 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
- Added 5 μL of NaOAc, 1.5 μL of glycogen and 150 μL of 100% ethanol.
- Left it at -80℃ for 1 hr.
- Centrifuged at 15,000 rpm for 15 min at 4℃.
- Removed supernatant and added 220 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 10 min at 4℃.
- Removed supernatant and air-dried at room temperature with light shield.
- 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
FastGeneTM 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
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
Ethanol Precipitation
Fujita, Ono
XbaⅠ - Thanatin - SpeⅠ
- Added 20 μL of NaOAc, 1.5 μL of glycogen and 600 μL of 100% ethanol.
- Left it at -80℃ for 1 hr.
- Centrifuged at 15,000 rpm for 15 min at 4℃.
- Removed supernatant and added 100 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 10 min at 4℃.
- Removed supernatant and air-dried at room temperature with light shield.
- 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)
- Added 3 μL of NaOAc, 1.5 μL of glycogen and 90 μL of 100% ethanol.
- Left it at -80℃ for 10 min.
- Centrifuged at 15,000 rpm for 5 min at 4℃.
- Removed supernatant and added 100 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 5 min at 4℃.
- Removed supernatant and air-dried at room temperature with light shield.
- 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
- Added 5 μL of NaOAc, 1.5 μL of glycogen and 150 μL of 100% ethanol.
- Left it at -80℃ for 1 hr.
- Centrifuged at 15,000 rpm for 15 min at 4℃.
- Removed supernatant and added 100 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 10 min at 4℃.
- Removed supernatant and air-dried at room temperature with light shield.
- 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)
- Added 5 μL of NaOAc, 1.5 μL of glycogen and 280 μL of 100% ethanol.
- Left it at -80℃ for 1 hr.
- Centrifuged at 15,000 rpm for 15 min at 4℃.
- Removed supernatant and added 100 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 10 min at 4℃.
- Removed supernatant and air-dried at room temperature with light shield.
- 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)
FastGeneTM 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)
FastGeneTM Gel Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
September
2015/09/01
Gel Extract
Nishimura
BBa_B0032, BBa_B0033, BBa_B0034, BBa_B0015, BBa_I0500
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
Mini-prep
Nishimura
BBa_R0010 on pSB1C3, BBa_I0500 on pSB1C3
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol
Electrophoresis
Nishimura
BBa_R0010 - BBa_B0034 on pSB1C3(dephosphorylated product), BBa_R0010 - BBa_B0034 on pSB1C3 (Gel extract product)
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
2% | 100 V | 60 min | 1/2 x TBE |
Ligation
Fujita, Nishimura
BBa_R0010 - BBa_B0034 on pSB1C3 / XbaⅠ - Thanatin - SpeⅠ
Reagent | Volume |
---|---|
BBa_R0010 - BBa_B0034 on pSB1C3 | 9.5 μL |
XbaⅠ - Thanatin - SpeⅠ | 0.5 μL |
Mighty Mix | 10 μL |
Total | 20 μL |
Ligation
Step | Temp. | Time | Process |
---|---|---|---|
1 | 16℃ | 30 min | Ligation |
Store | 4℃ | Hold | Store |
Ligation
Fujita
Ag43 on pSB1C3 / BamHⅠ - Thanatin - BglⅡ
Reagent | Volume |
---|---|
BBa_R0010 - BBa_B0034 on pSB1C3 | 9.5 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 |
Ethanol Precipitation
Fujita
Thanatin - Ag43 on pSB1C3
- Added 2 μL of NaOAc, 1.5 μL of glycogen and 60 μL of 100% ethanol.
- Left it at -80℃ for 1 hr.
- Centrifuged at 15,000 rpm for 15 min at 4℃.
- Removed supernatant and added 100 μL of 70% ethanol.
- Centrifuged at 15,000 rpm for 10 min at 4℃.
- Removed supernatant and air-dried at room temperature with light shield.
- Suspended with 10 μL of TE.
Transformation
Nishimura, Toyooka
BBa_I0500 - BBa_B0033 on pSB1C3,
- Added 5 μL of BBa_B0033 on pSB1C3, BBa_R0040, BBa_I0500 BBa_B0032 on pSB1C3, BBa_I0500 BBa_B0033 on pSB1C3 to 50 μL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 μL of the culture onto plate with LBC.
- Incubated the plate at 37℃ for 16 hours.
Digestion
Nishimura, Onoda
HLA, HLZ, BLA, BLZ, BBa_B0030
Reagent | Volume |
---|---|
HLA, HLZ, BLA, BLZ, BBa_B0030 | 10 μL |
SpeⅠ | 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 |
Electrophoresis
Nishimura
HLA, HLZ, BLA, BLZ
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
2% | 100 V | 60 min | 1/2 x TBE |
Gel Extract
Nishimura, Sakai, Ito, Kusumi
HLA, HLZ, BLA, BLZ
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
Ligation
Fujita, Sakai, Nishimura
BBa_B0015 on pSB1C3 / HLA, BLA, BLZ
Reagent | Volume |
---|---|
BBa_B0015 on pSB1C3 | 10 μL |
HLA , BLA, BLZ | 30 μL |
T4 Ligase | 4.5 μL |
10 × T4 DNA Ligase Buffer | 5 μL |
DW | 0.5 μL |
Total | 50 μL |
Ligation
Step | Temp. | Time | Process |
---|---|---|---|
1 | 16℃ | 30 min | Ligation |
Store | 4℃ | Hold | Store |
Ligation
Onoda, Nishimura, Fujita, Sakai
HLZ / BBa_B0015 on pSB1C3
Reagent | Volume |
---|---|
BBa_B0015 on pSB1C3 | 10 μL |
HLZ | 20 μL |
T4 Ligase | 3.5 μL |
10 × T4 DNA Ligase Buffer | 5 μL |
DW | 1.5 μL |
Total | 50 μL |
Ligation
Step | Temp. | Time | Process |
---|---|---|---|
1 | 16℃ | 30 min | Ligation |
Store | 4℃ | Hold | Store |
Electrophoresis
Sakai
HLA, HLZ, BLA, BLZ
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
2% | 50 V | 60 min | 1/2x TBE |
Ligation
Sakai
BBa_R0010 - BBa_B0034 on pSB1C3 / XbaⅠ - Thanatin - SpeⅠ (Digestion product)
Reagent | Volume |
---|---|
BBa_R0100 - BBa_B0034 on pSB1C3 | 15 μL |
XbaⅠ - Thanatin - SpeⅠ | 1.0 μL |
T4 ligase | 1.6 μL |
10 X T4 DNA Ligase Buffer | 2.0 μL |
DW | 0.4 μL |
Total | 20 μL |
Ligation
Step | Temp. | Time | Process |
---|---|---|---|
1 | 16℃ | 30 min | Ligation |
Store | 4℃ | Hold | Store |
Transformation
Sakai
BBa_R0010 - BBa_B0034 - XbaⅠ - Thanatin -SpeⅠ on pSB1C3
- Added 1.0 μL of BBa_R0010 - BBa_B0034 - XbaⅠ - Thanatin -SpeⅠ on pSB1C3 to 50 μL of thawed competent cells (DH5a) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 μL of the culture onto plate with LB C.
- Incubated the plate at 37℃ for 2 hours.
PCR
Ono
ABF-2, BLA, Thanatin, 10 × His - TEV - Thanatin, BLZ, HLZ
Reagent | Volume |
---|---|
ABF-2, BLA, Thanatin, 10 × His - TEV - Thanatin, BLZ, HLZ | 1 μL |
EX - F - Universal 10 μM | 1 μL |
PS - R 10 μM | 1 μL |
KOD - Plus - Neo | 1 μL |
10x 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 |
2015/09/02
Gel Extract
Mimata
EcoRⅠ - BBa_B0032 - XbaⅠ, EcoRⅠ - BBa_B0033 - XbaⅠ, EcoRⅠ - BBa_B0034 - XbaⅠ, SpeⅠ - BBa_B0015 - PstⅠ, EcoRⅠ - BBa_I0500 - SpeⅠ
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
Transformation
Nishimura
HLA, BLA, HLZ, BLZ
- Added 1 μL of HLA, BLA, HLZ, BLZ to 10 μL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 μL of the culture onto plate with LBC.
- Incubated the plate at 37℃ for 16 hours.
Colony PCR
Nishimura, Ono, Onoda, Mimata
BBa_R0010 - BBa_B0034 - Thanatin
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 |
Colony PCR
Nishimura, Ono, Onoda, Mimata
BBa_R0010 - BBa_B0034 - Thanatin
Reagent | Volume |
---|---|
Single Colony | - |
100UP - EX - F 10 μM | 0.4 μL |
SpeⅠ - Thanatin Rv 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
Nishimura, Ono, Onoda, Mimata
Ag43 - Thanatin
Reagent | Volume |
---|---|
Single Colony | - |
AGSP - BamHⅠ - Spindorin 10 μM | 0.4 μL |
BglⅡ - D - Thanatin Rv 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℃ | 40 sec | Elongation | 40 cycle |
Finish | 72℃ | 120 sec | Final Elongation | |
Store | 4℃ | Hold | Store |
Colony PCR
Nishimura, Ono, Onoda, Mimata
Ag43 - Thanatin
Reagent | Volume |
---|---|
Single Colony | - |
AGSP - BamHⅠ - Spindorin 10 μM | 0.4 μL |
Ag43 - bunit - Rv 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℃ | 40 sec | Elongation | 40 cycle |
Finish | 72℃ | 120 sec | Final Elongation | |
Store | 4℃ | Hold | Store |
Colony PCR
Nishimura, Ono, Onoda, Mimata
BBa_B0031 on pSB1A2
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
Nishimura, Ono, Onoda
BBa_R0010 - BBa_B0034 - Thanatin (colony PCR product), Ag43 Thanatin (colony PCR product), BBa_B0031 on pSB1C3(colony PCR product)
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
1% | 100 V | 50 min | 1/2 x TBE |
Electrophoresis
Nishimura, Ono, Mimata
BBa_I0500
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
1% | 100 V | 50 min | 1/2 x TBE |
Digestion
Nisimura, Ono, Mimata
BLZ, HLZ, ABF-2
Reagent | Volume |
---|---|
BLZ, HLZ, ABF-2 | 30 μL |
SpeⅠ | 1 μL |
Total | 31 μL |
Digestion
Step | Temp. | Time | Process |
---|---|---|---|
1 | 37℃ | 60 min | Digestion |
Store | 4℃ | Hold | Store |
Electrophoresis
Nishimura, Ono, Mimata
BLZ, HLZ, ABF-2,
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
2% | 100 V | 60 min | 1/2 x TBE |
Transformation
Mimata
HLA on pSB1C3, HLZ on pSB1C3, BLA on pSB1C3, BLZ on pSB1C3, BBa_B0015 on pSB1C3
- Added 1 μL of HLA on pSB1C3, HLZ on pSB1C3, BLA on pSB1C3, BLZ on pSB1C3, BBa_B0015 on pSB1C3 to 50 μL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 2000 μL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 μL of the culture onto plate with LBC.
- Incubated the plate at 37℃ for 16 hours.
2015/09/03
Ligation
Nishimura
BBa_B0015 on pSB1C3 / HLZ
Reagent | Volume |
---|---|
BBa_B0015 on pSB1C3 | 6 μL |
HLZ | 8 μL |
Mighty Mix | 14 μL |
Total | 28 μL |
Ligation
Step | Temp. | Time | Process |
---|---|---|---|
1 | 16℃ | 30 min | Ligation |
Store | 4℃ | Hold | Store |
Ligation
Nishimura
BBa_B0015 on pSB1C3 / HLA, BLA, BLZ
Reagent | Volume |
---|---|
BBa_B0015 on pSB1C3 | 6 μL |
HLA, BLA, BLZ | 14 μL |
Mighty Mix | 20 μL |
Total | 40 μL |
Ligation
Step | Temp. | Time | Process |
---|---|---|---|
1 | 16℃ | 30 min | Ligation |
Store | 4℃ | Hold | Store |
Dephosphorylation
Nishimura
BBa_B0015 on pSB1C3
Reagent | Volume |
---|---|
BBa_B0015 on pSB1C3 | 40 μL |
Antarctic Phosphatase | 4 μL |
Antarctic Phosphatase Buffer | 8 μL |
DW | 28 μL |
Total | 80 μL |
Dephosphorylation
Step | Temp. | Time | Process |
---|---|---|---|
1 | 37℃ | 30 min | Dephosphorylation |
2 | 65℃ | 10 min | Inactivation |
Store | 4℃ | Hold | Store |
Colony PCR
Nishimura, Ono, Fujita
BBa_R0010 - BBa_B0034 - Thanatin
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 |
Colony PCR
Nishimura, Ono, Fujita
BBa_R0010 - BBa_B0034 - Thanatin
Reagent | Volume |
---|---|
Single Colony | - |
100UP - EX - F 10 μM | 0.4 μL |
SpeⅠ - Thanatin - Rv 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
Nishimura, Ono, Fujita
Ag43 - Thanatin
Reagent | Volume |
---|---|
Single Colony | - |
AGSP - BamHⅠ - Spindorin 10 μM | 0.4 μL |
BglⅡ - D - Thanatin - Rv 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℃ | 40 sec | Elongation | 40 cycle |
Finish | 72℃ | 120 sec | Final Elongation | |
Store | 4℃ | Hold | Store |
Colony PCR
Nishimura, Ono, Fujita
Ag43 - Thanatin
Reagent | Volume |
---|---|
Single Colony | - |
AGSP - BamHⅠ - Spindorin 10 μM | 0.4 μL |
Ag43 - bunit - Rv 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℃ | 40 sec | Elongation | 40 cycle |
Finish | 72℃ | 120 sec | Final Elongation | |
Store | 4℃ | Hold | Store |
Colony PCR
Nishimura, Ono, Fujita
BBa_B0031 on pSB1A2
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
Nishimura, Ono, Fujita
BBa_R0010 - BBa_B0034 - Thanatin (colony PCR product), Ag43 Thanatin (colony PCR product), BBa_B0031 on pSB1C3(colony PCR product)
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
1% | 100 V | 50 min | 1/2 x TBE |
Transformation
Nishimura, Fujita
HLA, BLA, HLZ, BLZ
- Added 40 μL of HLA, BLA, HLZ, BLZ to 1 μL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 μL of the culture onto plate with LBchloramphenicol.
- Incubated the plate at 37℃ for 16 hours.
Mini-prep
Ono, Fujita
Ag43 - Thanatin on pSB1C3
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol
Digestion
Onoda
EcoRⅠ - BBa_R0010 - SpeⅠ
Reagent | Volume |
---|---|
EcoRⅠ - BBa_R0010 - SpeⅠ | 28.5 μL |
EcoRⅠ | 1.5 μL |
SpeⅠ | 1.5 μL |
10 × H Buffer | 3.5 μL |
Total | 35 μL |
Digestion
Step | Temp. | Time | Process |
---|---|---|---|
1 | 37℃ | 120 min | Digestion |
Store | 4℃ | Hold | Store |