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 hrs.
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 |
KAPA Taq | 10 µL |
DW | 8.4 µL |
Total | 20 µL |
2 Step Cycle (Tm value ≥ 63℃)
Step | Temp. | Time | Process | Cycle |
---|---|---|---|---|
Start | 94℃ | 120 sec | Initialization | |
Cycle 1 | 98℃ | 10 sec | Denaturation | 35 cycle |
Cycle 2 | 68℃ | 60 sec | Annealing / Elongation | 35 cycle |
Finish | 68℃ | 60 sec | Final Elongation | |
Store | 4℃ | Hold | Store |
Electrophoresis
Sakurai
BBa_K1524100
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
1% | 100 V | 30 min | 1/2 x TBE |
Liquid Culture
Sakurai
BBa_K1524100
Reagent | Volume |
---|---|
Single Colony | - |
LB | 2 µL |
Cultured for 16 hrs.
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 |
KAPA Taq | 5 µL |
DW | 4.2 µL |
Total | 10 µL |
2 Step Cycle (Tm value ≥ 63℃)
Step | Temp. | Time | Process | Cycle |
---|---|---|---|---|
Start | 94℃ | 120 sec | Initialization | |
Cycle 1 | 98℃ | 10 sec | Denaturation | 35 cycle |
Cycle 2 | 68℃ | 60 sec | Annealing / Elongation | 35 cycle |
Finish | 68℃ | 60 sec | Final Elongation | |
Store | 4℃ | Hold | Store |
Electrophoresis
Sakurai
BBa_K1524100
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
1% | 100 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 |
KAPA Taq | 5 µL |
DW | 4.2 µL |
Total | 10 µL |
2 Step Cycle (Tm value ≥ 63℃)
Step | Temp. | Time | Process | Cycle |
---|---|---|---|---|
Start | 94℃ | 120 sec | Initialization | |
Cycle 1 | 98℃ | 10 sec | Denaturation | 35 cycle |
Cycle 2 | 68℃ | 60 sec | Annealing / Elongation | 35 cycle |
Finish | 68℃ | 60 sec | Final Elongation | |
Store | 4℃ | Hold | Store |
Colony PCR
Sakurai
BBa_K1524100
Reagent | Volume |
---|---|
Single Colony | - |
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 |
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 (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 hrs.
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 hrs.
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 hrs.
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 hrs.
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 |
100UP- EX - F 10 µM | 1 µL |
200DN - PS - R 10 µM | 1 µL |
KOD - FX - Neo | 1 µL |
2 x 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 |
100UP - EX - F 10 µM | 1 µL |
200DN - PS - R 10 µM | 1 µL |
KOD - FX - Neo | 1 µL |
2 x 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 |
100UP - EX - F 10 µM | 1 µL |
200DN - PS - R 10 µM | 1 µL |
KOD - Plus - Neo | 1 µL |
10 x Buffer for KOD - Plus - 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 |
100UP - EX - F 10 µM | 1 µL |
200DN - PS - R 10 µM | 1 µL |
KOD - Plus - Neo | 1 µL |
10 x 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℃ | 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℃ | 60 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 |
Finish | 72℃ | 60 sec | Final Elongation | |
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 hrs.
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 hrs.
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 hrs.
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 hrs.
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 hrs.
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 hrs.
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 | 1/2 x 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 hrs.
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 - 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 - 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 - 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 - 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 |
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 | - | 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 |
10 x 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 |
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 |
2 × 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℃ | 30 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℃ | 30 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 | 1|
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 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 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 - XbaⅠ / SpeⅠ 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 (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.
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 | 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
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 | 1/2 x 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 (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 2 hours.
PCR
Ono
ABF-2, BLA, Thanatin, 10 × His tag - TEV - Thanatin, BLZ, HLZ
Reagent | Volume |
---|---|
ABF-2, BLA, Thanatin, 10 × His tag - TEV - Thanatin, BLZ, HLZ | 1 µL |
EX - F - Universal 10 µM | 1 µL |
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 | 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 LBC.
- 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 |
2015/09/04
Electrophoresis
Nishimura
ABF-2, BLA, Thanatin, 10 × His tag - TEV - Thanatin, BLZ, HLZ, ABF-2
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
2% | 100 V | 60 min | 1/2 x TBE |
Mini-prep
Nishimura, Ono
Ag43 - thanatin
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol
Sequencing
Nishimura
thanatin - Ag43
Reagent | Volume |
---|---|
Ag43 thanatin | 1 µL |
BBa_I0500 - Fw, Ag43 - Rv | 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 | - | 25 cycle |
Cycle 2 | 60℃ | 240 sec | - | 25 cycle |
Store | 4℃ | Hold | Store |
Sequencing
Nishimura
BBa_I0500 - BBa_B0032, BBa_I0500 - BBa_B0033, BBa_I0500 - BBa_B0034
Reagent | Volume |
---|---|
100UP - EX - F | 1 µL |
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 | - | 25 cycle |
Cycle 2 | 60℃ | 240 sec | - | 25 cycle |
Store | 4℃ | Hold | Store |
Ethanol Precipitation
Nishimura, Fujita, Mimata
Ag43 - thanatin, BBa_I0500 - BBa_B0032, BBa_I0500 - BBa_B0033, BBa_I0500 - BBa_B0034
- Added 1 µL of NaOAc, 1.5 µL of glycogen and 30 µL of 100% ethanol.
- Left it at -80℃ for 10 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 HiDi.
- Added 5 µL of BLZ - BBa_B0015, HLZ - BBa_B0015, ABF-2 - BBa_B0015, BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015, BBa_R0010 - BBa_B0034 - BLA - BBa_B0015 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 18 hours.
Transformation
Fujita, Mimata
BLZ - BBa_B0015, HLZ - BBa_B0015, ABF-2 - BBa_B0015, BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015, BBa_R0010 - BBa_B0034 - BLA - BBa_B0015
Digestion
Fujita, Mimata
pSB1C3
Reagent | Volume |
---|---|
pSB1C3 | 5 µL |
EcoRⅠ | 1 µL |
PstⅠ | 1 µL |
10 × H Buffer | 5 µL |
DW | 38 µL |
Total | 50 µL |
Digestion
Step | Temp. | Time | Process |
---|---|---|---|
1 | 37℃ | 120 min | Digestion |
2 | 80℃ | 20 min | Inactivation |
Store | 4℃ | Hold | Store |
Digestion
Fujita, Mimata
pSB1C3
Reagent | Volume |
---|---|
pSB1C3 | 5 µL |
XbaⅠ | 1 µL |
SpeⅠ | 1 µL |
CutSmart Buffer | 5 µL |
DW | 38 µL |
Total | 50 µL |
Digestion
Step | Temp. | Time | Process |
---|---|---|---|
1 | 37℃ | 120 min | Digestion |
2 | 80℃ | 20 min | Inactivation |
Store | 4℃ | Hold | Store |
Digestion
Fujita, Mimata
ABF-2, Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix, 10 x His tag - TEV - Thanatin
Reagent | Volume |
---|---|
ABF-2, Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix, 10 x His tag - TEV - Thanatin | 20 µL |
EcoRⅠ | 1 µL |
PstⅠ | 1 µL |
10 x H Buffer | 5 µL |
DW | 23 µL |
Total | 50 µL |
Digestion
Step | Temp. | Time | Process |
---|---|---|---|
1 | 37℃ | 120 min | Digestion |
2 | 80℃ | 20 min | Inactivation |
Store | 4℃ | Hold | Store |
Digestion
Fujita, Mimata
BLA
Reagent | Volume |
---|---|
BLA | 20 µL |
XbaⅠ | 1 µL |
PstⅠ | 1 µL |
CutSmart Buffer | 5 µL |
DW | 23 µL |
Total | 50 µL |
Digestion
Step | Temp. | Time | Process |
---|---|---|---|
1 | 37℃ | 120 min | Digestion |
2 | 80℃ | 20 min | Inactivation |
Store | 4℃ | Hold | Store |
Digestion
Fujita, Mimata
BLZ, HLZ, ABF-2
Reagent | Volume |
---|---|
BLZ, HLZ, ABF-2 | 20 µL |
EcoRⅠ | 1 µL |
SpeⅠ | 1 µL |
10 x H Buffer | 5 µL |
DW | 23 µL |
Total | 50 µL |
Digestion
Step | Temp. | Time | Process |
---|---|---|---|
1 | 37℃ | 120 min | Digestion |
2 | 80℃ | 20 min | Inactivation |
Store | 4℃ | Hold | Store |
2015/09/05
Colony PCR
Fujita, Mimata
BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3
Reagent | Volume |
---|---|
Single Colony | - |
100UP - EX - F 10 µM | 0.4 µL |
200DN - PS - R 10 µM | 0.4 µL |
KAPA Taq | 5.0 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 | 35 cycle |
Cycle 2 | 57.6℃ | 30 sec | Annealing | 35 cycle |
Cycle 3 | 72℃ | 210 sec | Elongation | 35 cycle |
Finish | 72℃ | 120 sec | Final Elongation | |
Store | 4℃ | Hold | Store |
Electrophoresis
Fujita, Mimata
BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3 Mini-prep Mimata BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - 10 x His tag - TEV - Thanatin - BBa_B0015, BBa_R0010 - BBa_B0034 - Thanatin - BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 - ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3
Ligation Mimata pSB1C3 / Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix Ligation Ligation Mimata pSB1C3 / Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 Ligation Electrophoresis Mimata Thanatin on pSB1C3, TEV - Thanatin on pSB1C3 Digestion Sakai BBa_I0500 - BBa_B0034, BBa_I0500 - BBa_B0033, BBa_I0500 - BBa_B0032 Digestion Digestion Sakai Ag43 - Thanatin Digestion 2015/09/06 Colony PCR Fujita BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3 3 Step Cycle (Tm value ≤ 63℃) Electrophoresis Fujita BLZ - BBa_B0015 on pSB1C3, HLZ - BBa_B0015 on pSB1C3, ABF-2 - BBa_B0015 on pSB1C3, BBa_I0500 - BBa_B0034 - BLA - BBa_B0015 on pSB1C3 (Colony PCR product)
Gel Concentration Voltage Time Buffer 1% V 30 min 1/2 x TBE
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol
Reagent Volume pSB1C3 2.5 µL Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - Thanatin - BBa_B0015 - Suffix 40 µL Mighty Mix 42.5 µL Total 85 µL
Step Temp. Time Process 1 16℃ 30 min Ligation 2 70℃ 10 min Inactivation Store 4℃ Hold Store
Reagent Volume pSB1C3 2.5 µL Prefix - BBa_R0010 - BBa_B0034 - XbaⅠ / SpeⅠ scar - 10 × His tag - TEV - Thanatin - BBa_B0015 40 µL Mighty Mix 42.5 µL Total 85 µL
Step Temp. Time Process 1 16℃ 30 min Ligation 2 70℃ 10 min Inactivation Store 4℃ Hold Store
Gel Concentration Voltage Time Buffer 1% 100 V 60 min 1/2 x TBE
Reagent Volume BBa_I0500 - BBa_B0034, BBa_I0500 - BBa_B0033, BBa_I0500 - BBa_B0032 20 µL PstⅠ 1.5 µL SpeⅠ 1.5 µL 10 x H Buffer 3 µL DW 4 µL Total 30 µL
Step Temp. Time Process 1 37℃ 120 min Digestion 2 80℃ 20 min Inactivation Store 4℃ Hold Store
Reagent Volume Ag43 - Thanatin 20 µL BglⅡ 2.0 µL CutSmart Buffer 3 µL DW 5 µL Total 30 µL
Step Temp. Time Process 1 37℃ 120 min Digestion 2 80℃ 20 min Inactivation Store 4℃ Hold Store
Reagent Volume Single Colony - 100UP - EX - F 10 µM 0.4 µL 200DN - PS - R 10 µM 0.4 µL KAPA Taq 5.0 µL DW 4.2 µL Total 10 µL
Step Temp. Time Process Cycle Start 95℃ 120 sec Initialization Cycle 1 95℃ 30 sec Denaturation 35 cycle Cycle 2 57.6℃ 30 sec Annealing 35 cycle Cycle 3 72℃ 210 sec Elongation 35 cycle Finish 72℃ 120 sec Final Elongation Store 4℃ Hold Store
Gel Concentration Voltage Time Buffer 1% 100 V 30 min 1/2 x TBE