Team:HokkaidoU Japan/Notebook/ecoli

Notebook

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E. coli

January

2015/01/21

Transformation

Sakurai

BBa_K1524100

  1. Added 5 μL of antiBBa_E1010 on BBa_K1524100 to 20 μL of thawed competent cells (DH5α) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Spread 300 μL of the culture onto plate with LBA.
  5. Incubate 2ml regent with ampicillin at 37℃ for 20 hours.

2015/01/22

Colony PCR

Sakurai

BBa_K1524100

ReagentVolume
Single Colony-
100UP - EX - F 10 μM0.8 μL
XhoⅠ - RBS - NcoⅠ 10 μM0.8 μL
TAKA Taq10 μL
DW8.4 μL
Total20 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Finish68℃60 secFinal Elongation
Store4℃HoldStore

Electrophoresis

Sakurai

BBa_K1524100

Gel ConcentrationVoltageTimeBuffer
1%100 V30 min1/2 x TBE

Liquid Culture

Sakurai

BBa_K1524100

ReagentVolume
Single Colony-
LB2 μL

Cultured for 16 hours.

2015/01/26

Colony PCR

Sakurai

BBa_K1524100

ReagentVolume
Single Colony-
100UP - EX-F 10 μM0.4 μL
XhoⅠ - RBS - NcoⅠ 10 μM0.4 μL
TAKA Taq5 μL
DW4.2 μL
Total10 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Finish68℃60 secFinal Elongation
Store4℃HoldStore

Electrophoresis

Sakurai

BBa_K1524100

Gel ConcentrationVoltageTimeBuffer
1%100 V30 min1/2 x TBE

Colony PCR

Sakurai

BBa_K1524100

ReagentVolume
Single Colony-
100UP - EX - F 10 μM0.4 μL
XhoⅠ - RBS - NcoⅠ 10 μM0.4 μL
TAKA Taq5 μL
DW4.2 μL
Total10 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Finish68℃60 secFinal Elongation
Store4℃HoldStore

Colony PCR

Sakurai

BBa_K1524100

ReagentVolume
Single Colony-
EX - F - Universal 10 μM0.4 μL
PS - R 10 μM0.4 μL
TAKA Taq5 μL
DW4.2 μL
Total10 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Finish68℃60 secFinal Elongation
Store4℃HoldStore

March

2015/03/10

Competent Cells

Tanaka,Sakurai

BL21 (DE3) pLysS

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

2015/03/11

PCR

Sakurai

BBa_R0011

ReagentVolume
BBa_R00111 μL
100UP - EX - F 10 μM1.5 μL
200DN - PS - R 10 μM1.5 μL
KOD - Plus - NEO1 μL
10 x PCR Buffer for KOD - Plus - NEO 5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW32 μL
Total50 μL

BBa_0030 - BBa_E1010

ReagentVolume
BBa_0030 - BBa_E10101 μL
100UP - EX - F 10 μM1.5 μL
200DN - PS - R 10 μM1.5 μL
KOD - Plus - NEO1 μL
10 x PCR Buffer for KOD - Plus - NEO5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW32 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 262.6℃30 secAnnealing30 cycle
Cycle 368℃1 minElongation30 cycle
Store4℃HoldStore

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

ReagentVolume
BBa_R001144 μL
XbaI1 μL
CutSmart Buffer5 μL
Total50 μL

BBa_0030 - BBa_E1010

ReagentVolume
BBa_0030 - BBa_E101044 μL
XbaI1 μL
CutSmart Buffer5 μL
Total50 μL

Digestion

StepTemp.TimeProcess
137℃300 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Electrophoresis

Sakurai

BBa_R0011, BBa_0030 - BBa_E1010

Gel ConcentrationVoltageTimeBuffer
1%100 V30 min1/2 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 ConcentrationVoltageTimeBuffer
1%100 V30 min1/2 x TBE

May

2015/05/13

Transformation

Onoda

pET15b

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

Transformation

Onoda, Sakurai

pET16b

  1. Added 1 μL of pET16b to 50 μL of thawed competent cells (DH5α) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Spread 50 μL of the culture onto plate with LBA.
  5. Incubated the plate at 37℃ for 16 hours.

Competent Cells

Onoda

Rosetta

  1. Thawed original competent cells (Rosetta) on ice.
  2. Added 5 μL of original competent cells to 2 mL of LB.
  3. Incubated the cells for 16 hrs at 37℃.
  4. Added 5 μL, 50 μL, and 500 μL of original cells to 100 mL of LB.
  5. Incubated the cells at 130 rpm for 24 hrs at 20℃, until OD600 reach 0.5.
  6. Took 50 mL of incubated cells to two differnt culture tubes and centrifuged them at 3,000 rpm for 20 min at 4℃.
  7. Removed supernatant and added 75 mL of TB to each tube.
  8. Brought them to a one tube and centrifuged at 3,000 rpm for 20 min at 4℃.
  9. Removed supernatant and added 32 mL of TB.
  10. Added 32 μL of DMSO 10 times.
  11. Took 50 μL and froze with liquid nitrogen.

2015/05/27

Transformation

Mimata, Onoda, Nishimura

BBa_E0040

  1. Added 1 μL of BBa_E0040 to thawed competent cells (Rosetta and DH5α) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 200 μL of LB.
  5. Spread 300 μL of the culture onto plate with LBA.
  6. Incubated the plate at 37℃ for 16 hours.

Transformation

Mimata, Onoda, Ono, Nishimura

mBBa_R0040

  1. Added 1 μL of mBBa_R0040 to thawed competent cells (Rosetta and DH5α) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 200 μL of LB.
  5. Spread 300 μL of the culture onto plate with LBA.
  6. Incubated the plate at 37℃ for 16 hours.

2015/05/29

Mini-prep

Mimata, Onoda, Ono, Nishimura

BBa_E0040, mBBa_R0040
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

PCR

Onoda, Ono

BBa_E0040

ReagentVolume
BBa_E00401 μL
EX - F - Universal 10 μM1 μL
200DN - PS - R 10 μM1 μL
KOD - FX - Neo1 μL
10x PCR Buffer for KOD - FX - Neo 5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

mBBa_R0040

ReagentVolume
mBBa_R00401 μL
EX - F - Universal 10 μM1 μL
200DN - PS - R 10 μM1 μL
KOD - FX - Neo1 μL
10x PCR Buffer for KOD - FX - Neo 5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycles
Cycle 268℃60 secAnnealing / Elongation30 cycles
Store4℃HoldStore

2015/05/30

Electrophoresis

Mimata, Onoda, Ono, Nishimura

BBa_E0040, mBBa_R0040

Gel ConcentrationVoltageTimeBuffer
2%100 V30 min1/2 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

ReagentVolume
BBa_E004020 μL
DW5 μL
SpeⅠ1 μL
EcoRⅠ1 μL
CutSmart Buffer3 μL
Total30 μL

mBBa_R0040

ReagentVolume
mBBa_R004020 μL
DW5 μL
SpeⅠ1 μL
EcoRⅠ1 μL
CutSmart Buffer3 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Digestion

Onoda, Ono

pET15b

ReagentVolume
pET15b10 μL
DW6 μL
SpeⅠ1 μL
EcoRⅠ1 μL
CutSmart Buffer2 μL
Total20 μL

pET16b

ReagentVolume
pET16b10 μL
DW6 μL
SpeⅠ1 μL
EcoRⅠ1 μL
CutSmart Buffer2 μL
Total20 μL

pSB1A3

ReagentVolume
pSB1A310 μL
DW6 μL
SpeⅠ1 μL
EcoRⅠ1 μL
CutSmart Buffer2 μL
Total20 μL

pSB4C5

ReagentVolume
pSB4C52 μL
DW14 μL
SpeⅠ1 μL
EcoRⅠ1 μL
CutSmart Buffer2 μL
Total20 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

2015/05/31

Electrophoresis

Mimata, Onoda, Ono, Nishimura

BBa_E0040, BBa_R0040, pET15b, pET16b, pSB1A3, pSB4C5

Gel ConcentrationVoltageTimeBuffer
1%100 V30 min1/2 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 ConcentrationVoltageTimeBuffer
1%100 V30 min1/2 x TBE

PCR

Mimata, Onoda

BBa_E0040

ReagentVolume
BBa_E00401 μL
EX - F - Universal 10 μM1 μL
200DN - PS - R 10 μM1 μL
KOD Plus NEO1 μL
KOD Plus NEO 10x Buffer5 μL
2 mM dNTPS5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

mBBa_R0040

ReagentVolume
mBBa_R00401 μL
EX - F - Universal 10 μM1 μL
200DN - PS - R 10 μM1 μL
KOD Plus NEO1 μL
KOD Plus NEO 10x Buffer5 μL
2 mM dNTPS5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃60 secAnnealing / Elongation30 cycle
Store4℃HoldStore

June

2015/06/10

Digestion

Mimata, Onoda

BBa_E0040

ReagentVolume
BBa_E004016 μL
SpeⅠ1 μL
EcoRⅠ1 μL
CutSmart Buffer2 μL
Total20 μL

mBBa_R0040

ReagentVolume
mBBa_R004016 μL
SpeⅠ1 μL
EcoRⅠ1 μL
CutSmart Buffer2 μL
Total20 μL

Digestion

StepTemp.TimeProcess
137℃600 minDigestion
260℃15 minInactivation
Store4℃HoldStore

2015/06/16

PCR

Mimata

thanatin fragment for TA cloning

ReagentVolume
TA - F - primer1 μL
TA - R - primer1 μL
KAPA Taq25 μL
DW23 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃180 secInitialization
Cycle 195℃30 secDenaturation35 cycle
Cycle 263℃30 secAnnealing35 cycle
Cycle 372℃10 secElongation35 cycle
72℃60 sec
Store4℃HoldStore

2015/06/17

Electrophoresis

Mimata

thanatin fragment for TA cloning

Gel ConcentrationVoltageTimeBuffer
2%100 V30 min1/2 x TBE

Annealing Oligos and Elongation

Ito

thanatin fragment for TA cloning

ReagentVolume
TA - F - primer 1 μM1 μL
TA - R - primer 1 μM1 μL
KAPA Taq25 μL
DW23 μL
Total50 μL

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start95℃1 minInitialization
Cycle195℃ to 23℃45 minAnnealing1 cycle
Cycle 272℃1 minElongation1 cycle
Store4℃HoldStore

2015/06/19

Electrophoresis

Ito

thanatin fragment for TA cloning

Gel ConcentrationVoltageTimeBuffer
2%100 V30min1/2 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

ReagentVolume
pGEM - T vector1.7μL
Thanatin fragment for TA cloning0.15μL
Mighty Mix1.85μL
T4 Ligase0.18μL
DW6.12μL
Total10μL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
265℃10 minInactivation
Store4℃HoldStore

2015/06/21

Transformation

Ito

pGEM - T vector

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

July

2015/07/25

Transformation

Onoda

BBa_B0015 on pSB1C3

  1. Added 1 μL of BBa_B0015 on pSB1C3 to 50 μL of thawed competent cells (DH5α Turbo) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 500 μL of LB.
  5. Incubated the cells for 2 hrs at 37℃.
  6. Spread 300 μL of the culture onto plate with LBC.
  7. Incubated the plate at 37℃ for 16 hours.

Transformation

Onoda

BBa_R0010 - BBa_B0034 on pSB1C3

  1. Added 1 μL of BBa_R0010 - BBa_B0034 on pSB1C3 to 50 μL of thawed competent cells (DH5α Turbo) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 500 μL of LB.
  5. Incubated the cells for 2 hrs at 37℃.
  6. Spread 300 μL of the culture onto plate with LBC.
  7. Incubated the plate at 37℃ for 16 hours.

Transformation

Onoda

BBa_I0500 - BBa_B0034 on pSB1C3

  1. Added 1 μL of BBa_I0500 - BBa_B0034 on pSB1C3 to 50 μL of thawed competent cells (DH5α Turbo) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 500 μL of LB.
  5. Incubated the cells for 2 hrs at 37℃.
  6. Spread 300 μL of the culture onto plate with LBC.
  7. Incubated the plate at 37℃ for 16 hours.

Transformation

Onoda

BBa_B0034 on pSB1A2

  1. Added 1 μL of BBa_B0034 on pSB1A2 to 50 μL of thawed competent cells (DH5α Turbo) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 500 μL of LB.
  5. Spread 300 μL of the culture onto plate with LBA.
  6. Incubated the plate at 37℃ for 16 hours.

PCR

Onoda

BBa_B0015 on pSB1C3

ReagentVolume
BBa_B0015 on pSB1C31 μL
100UP - EX - F 10 μM1 μL
200DN - PS - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

BBa_R0010 - BBa_B0034 on pSB1C3

ReagentVolume
BBa_R0010 - BBa_B0034 on pSB1C31 μL
100UP - EX - F 10 μM1 μL
200DN - PS - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

BBa_I0500 - BBa_B0034 on pSB1C3

ReagentVolume
BBa_I0500 - BBa_B0034 on pSB1C31 μL
100UP - EX - F 10 μM1 μL
200DN - PS - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

BBa_B0034 on pSB1A2

ReagentVolume
BBa_B0034 on pSB1A21 μL
100UP - EX - F 10 μM1 μL
200DN - PS - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 262.6℃30 secAnnealing30 cycle
Cycle 368℃60 secElongation30 cycle
Store4℃HoldStore

Electrophoresis

Onoda

BBa_B0015 on pSB1C3, BBa_R0010 - BBa_B0034 on pSB1C3, BBa_I0500 - BBa_B0034 on pSB1C3, BBa_B0034 on pSB1A2

Gel ConcentrationVoltageTimeBuffer
2%100 V30 min1/2 x TBE

2015/07/26

Liquid Culture

Ono

BBa_I0500 - BBa_B0034 on pSB1A2

ReagentVolume
Single Colony-
LB2000 μL
Ampicillin2 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 ConcentrationVoltageTimeBuffer
2%100 V30 min1/2 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

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

2015/08/05

Colony PCR

Ito

pGEM T vector

ReagentVolume
Single Colony-
NdeⅠ - F - primer 10 μM0.4 μL
BamHⅠ - R - primer 10 μM0.4 μL
KAPA Taq5.0 μL
DW4.2 μL
Total10 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃30 secDenaturation35 cycle
Cycle 268℃20 secAnnealing / Elongation35 cycle
Store4℃HoldStore

Electrophoresis

Ito

NdeⅠ - Thanatin - BamHⅠ

Gel ConcentrationVoltageTimeBuffer
2%100V60 min1/2x TBE

Gel Extract

Ito

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

Ligation

Ito

pET vector / NdeⅠ - Thanatin - BamHⅠ

ReagentVolume
pET vector1 μL
NdeⅠ - Thanatin - BamHⅠ3 μL
10 × T4 DNA Ligase buffer5 μL
T4 Ligase1 μL
Total10 μL

Ligation

StepTemp.TimeProcess
14℃30 minLigation
265℃10 minInactivation
Store4℃HoldStore

Transformation

Ito

BBa_E1010 - BBa_B0034 - BBa_E0040 - BBa_B0015 on pSB1C3

  1. Added 1 μL of Thanatin on pET vector to 50 μL of thawed competent cells (Rosetta) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Spread 300 μL of the culture onto plate with LBA.
  5. Incubated the plate at 37℃ for 16 hours.

2015/08/10

Streaking (Single Colony Isolation)

Ito, Mimata, Mitsumoto, Onoda, Sakai

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

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

2015/08/11

Mini-prep

Ito, Mimata, Mitsumoto, Onoda, Sakai

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

Digestion

Ito, Mimata, Onoda, Sakai, Kusumi

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

ReagentVolume
pSB1C320 μL
DW23 μL
Bgl Ⅱ2 μL
3.1 Buffer5 μL
Total50 μL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
270℃15 minInactivation
Store4℃HoldStore

Electrophoresis

Ito, Mimata, Onoda, Sakai, Nishimura, Kusumi

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

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

2015/08/12

Gel Extract

Nishimura, Sakai

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

Electrophoresis

Ito, Sakai, Fujita

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

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 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)

ReagentVolume
pSB1C31 μL
T7 promoter primer / SP6 promoter primer1.5 μL
Ready Reaction Premix1 μL
5x Sequencing Buffer1.5 μL
DW5 μL
Total10 μL

Sequencing

StepTemp.TimeProcessCycle
Start96℃10 secDenaturation
Cycle 150℃5 sec-25 cycle
Cycle 260℃240 sec-25 cycle
Store4℃HoldStore

Ethanol Precipitation

Ito, Onoda, Nishimura, Fujita, Mimata

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

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

PCR

Ito, Onoda, Tanaka, Nishimura, Mimata

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

ReagentVolume
XbaⅠ - BBa_B0034 - XbaⅠ / SpeⅠ scar - Met - 10 × His tag - Thanatin - SpeⅠ1 μL
BamHⅠ - Thanatin forward Neo 10 μM1 μL
BglⅡ - Asp - Thanatin reverese Neo 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃20 secAnnealing / Elongation25 cycle
Store4℃HoldStore

Digestion

Mimata

NdeⅠ - Thanatin - BamHⅠ on pET vector

ReagentVolume
NdeⅠ - Thanatin - BamHⅠ on pET vector10 μL
NdeⅠ1 μL
BamHⅠ1 μL
CutSmart Buffer5 μL
DW33 μL
Total50 μL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
270℃15 minInactivation
Store4℃HoldStore

2015/08/14

PCR

Fujita, Nishimura, Onoda

Thanatin (Mini-prep product)

ReagentVolume
Thanatin fragment1 μL
T7 - promoter primer 10 μM1 μL
SP6 - promoter primer 10 μM1 μL
KOD - Plus - Neo1 μL
10× PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 66℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation25 cycle
Cycle 25010 secAnnealing25 cycle
Cycle 368℃30 secElongation25 cycle
Store4℃HoldStore

Electrophoresis

Fujita, Nishimura, Onoda, Mimata

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

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

Annealing of Oligonucleotides

Onoda, Nishimura, Fujita

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
TA - F - primer 10 μM1 μL
TA - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW34 μL
Total50 μL

Annealing of Oligonucleotides

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation10 cycle
Cycle 26010 secAnnealing10 cycle
Cycle 368℃30 secElongation10 cycle
Store4℃HoldStore

PCR

Nishimura, Onoda

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
Thanatin fragment (derived from annealing TA primers)1 μL
BamHⅠ - Thanatin Neo 10 μM1 μL
BglⅡ - Asp - thanatin Neo 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 66℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Store4℃HoldStore

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 ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

Transformation

Fujita, Mitsumoto

BBa_B0031 on pSB1A2, BBa_E0040 on pSB1A2

  1. Added 1 μL of BBa_B0031 on pSB1A2, BBa_E0040 on pSB1A2 to 20 μL of thawed competent cells (DH5α) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Spread 300 μL of the culture onto plate with LBA.
  5. Incubated the plate at 37℃ for 12 hours.

Transformation

Fujita, Mitsumoto

BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0030

  1. Added 1 μL of BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0030 to 20 μL of thawed competent cells (DH5α) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 200 μL of LB.
  5. Incubated the cells for 2 hrs at 37℃.
  6. Spread 300 μL of the culture onto plate with LBC.
  7. Incubated the plate at 37℃ for 12 hours.

PCR

Fujita, Mitsumoto

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

ReagentVolume
Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E00401 μL
100UP - EX - F 10 μM1 μL
200DN - PS - R 10 μM1 μL
KOD - Plus - Neo1 μL
10× PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Store4℃HoldStore

2015/08/15

Electrophoresis

Fujita, Nishimura

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

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

PCR

Fujita, Nishimura, Ono

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

ReagentVolume
BBa_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031, BBa_B0030, BBa_E00401 μL
100UP - EX - F 1 μM1 μL
200DN - PS - R 1 μM1 μL
KOD - Plus - Neo1 μL
10× PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Store4℃HoldStore

Electrophoresis

Fujita, Nishimura

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

PCR

Nishimura, Ono

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
Thanatin fragment (derived from annealing TA primers)1 μL
NdeⅠ - F - primer 10 μM1 μL
BamHⅠ - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10× PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃30 secAnnealing / Elongation25 cycle
Store4℃HoldStore

PCR

Sakai, Ono

Thanatin fragment (PCR 2STEP product)

ReagentVolume
Thanatin fragment (PCR 2STEP product)1 μL
BamHⅠ - Thanatin - F - Neo 10 μM1 μL
BglⅡ - Tanatin - R - Neo 10 μM1 μL
KOD - Plus - Neo1 μL
KOD Plus NEO 10 x Buffer5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃30 secAnnealing / Elongation25 cycle
Store4℃HoldStore

Annealing Oligos and Elongation

Onoda

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
TA - F - primer 10 μM1 μL
TA - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW34 μL
Total50 μL

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start95℃1 minInitialization
Cycle195℃ to 23℃45 minAnnealing1 cycle
Cycle 268℃30 secElongation1 cycle
Store4℃HoldStore

Electrophoresis

Ono, Onoda

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

Gel ConcentrationVoltageTimeBuffer
2%100 V30 min1/2 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)

ReagentVolume
Thanatin fragment (derived from annealing TA primers)1 μL
NdeI - F - primer 10 μM1 μL
BamHI - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃60 secAnnealing / Elongation25 cycle
Store4℃HoldStore

Electrophoresis

Nishimura, Ono

Thanatin fragment (PCR product)

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

Colony PCR

Onoda, Ono, Nishimura

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

ReagentVolume
Single Colony-
NdeI - F - primer 10 μM0.4 μL
BamHI - R - primer 10 μM0.4 μL
KAPA Taq5 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 262.930 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Onoda, Ono, Nishimura

BBa_B0031 on pSB1A2 into DH5α (as positive control)

ReagentVolume
Single Colony-
100UP - EX - F 10 μM0.4 μL
200DN - PS - R 10 μM0.4 μL
KAPA Taq5 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation30 cycle
Cycle 257.230 secAnnealing30 cycle
Cycle 372℃30 secElongation30 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Electrophoresis

Onoda, Ono

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

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

Electrophoresis

Ono, Mitsumoto, Fujita

BamHⅠ - Thanatin - BglⅡ, NdeⅠ - Thanatin - BamHⅠ

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

PCR

Onoda

Thanatin fragment (Mini-prep product)

ReagentVolume
Thanatin fragment (Mini-prep product)1 μL
BamHⅠ - Thanatin - F - Neo 10 μM1 μL
BglⅡ - Asp - Thanatin - R - Neo 10 μM1 μL
KOD - Plus - Neo1 μL
10× PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation45 cycle
Cycle 265.130 secAnnealing45 cycle
Cycle 368℃30 secElongation45 cycle
Store4℃HoldStore

PCR

Onoda

Thanatin fragment (Mini-prep product)

ReagentVolume
Thanatin fragment (Mini-prep product)1 μL
NdeⅠ - F - primer 10 μM1 μL
BamHⅠ - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10× PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation45 cycle
Cycle 266.530 secAnnealing45 cycle
Cycle 368℃30 secElongation45 cycle
Store4℃HoldStore

Electrophoresis

Onoda

Thanatin fragment for TA cloning and last PCR prduct

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

PCR

Fujita, Mimata

Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031

ReagentVolume
Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B00311 μL
100UP - EX - F 10 μM1 μL
200DN - PS - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃60 secAnnealing / Elongation35 cycle
Store4℃HoldStore

Electrophoresis

Fujita, Mimata

Ba_B0033, BBa_B0032, BBa_R0040, BBa_R0010, BBa_E1010, BBa_B0031

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Electrophoresis

Mitsumoto, Fujita

BamHI - Thanatin - BglⅡ, NdeI - Thanatin - BamHI

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Colony PCR

Ono, Onoda

Thanatin fragment on pGEM - T vector into DH5α

ReagentVolume
Single Colony-
NdeⅠ - F - primer 10 μM0.4 μL
BamHⅠ - R - primer 10 μM0.4 μL
KAPA Taq5 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 262.930 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Ono, Onoda

Thanatin fragment on pGEM - T vector into DH5α

ReagentVolume
Single Colony-
T7 promoter primer 10 μM0.4 μL
SP6 promoter primer 10 μM0.4 μL
KAPA Taq5 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25130 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Ono, Onoda

BBa_B0031 on pSB1A2 into DH5α (as positive control)

ReagentVolume
Single Colony-
100UP - EX - F 10 μM0.4 μL
200DN - PS - R 10 μM0.4 μL
KAPA Taq5 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Electrophoresis

Onoda

Thanatin fragment (Colony PCR product)

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
TA - F - primer 10 μM1 μL
TA - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW34 μL
Total50 μL

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start95℃1 minInitialization
Cycle195℃ to 23℃60 secAnnealing45 cycle
Cycle 268℃30 secElongation45 cycle
Store4℃HoldStore

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
TA - F - primer 10 μM1 μL
TA - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW34 μL
Total50 μL

Annealing Oligos and Elongation

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation10 cycle
Cycle 26010 secAnnealing10 cycle
Cycle 368℃30 secElongation10 cycle
Store4℃HoldStore

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
TA - F - primer 10 μM1 μL
TA - R - primer 10 μM1 μL
KOD - FX - Neo1 μL
10× PCR Buffer for KOD - FX - Neo25 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW14 μL
Total50 μL

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start95℃1 minInitialization
Cycle195℃ to 23℃60 secAnnealing45 cycle
Cycle 268℃30 secElongation45 cycle
Store4℃HoldStore

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
TA - F - primer 10 μM1 μL
TA - R - primer 10 μM1 μL
KOD - FX - NEO1 μL
10× PCR Buffer for KOD - FX - Neo25 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW14 μL
Total50 μL

Annealing Oligos and Elongation

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation10 cycle
Cycle 26010 secAnnealing10 cycle
Cycle 368℃30 secElongation10 cycle
Store4℃HoldStore

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
Single Colony-
100UP - EX - F 10 μM1 μL
200DN - PS - R 10 μM1 μL
KAPA Taq25 μL
DW23 μL
Total50 μL

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start95℃1 minInitialization
Cycle 195℃ to 23℃60 secAnnealing45 cycle
Cycle 268℃30 secElongation45 cycle
Store4℃HoldStore

Annealing Oligos and Elongation

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

ReagentVolume
Single Colony-
100UP - EX - F 10 μM1 μL
200DN - PS - R 10 μM1 μL
KAPA Taq25 μL
DW23 μL
Total50 μL

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation10 cycle
Cycle 26010 secAnnealing10 cycle
Cycle 368℃30 secElongation10 cycle
Store4℃HoldStore

Electrophoresis

Mitsumoto

Thanatin fragment (derived from annealing TA primers)

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

2015/08/17

Annealing Oligos and Elongation

Nishimura, Onoda

Thanatin fragment (Mini-prep product)

ReagentVolume
TA - F - primer 10 μM1 μL
TA - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW34 μL
Total50 μL

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation10 cycle
Cycle 26010 secAnnealing10 cycle
Cycle 368℃3030 secElongation10 cycle
Store4℃HoldStore

Annealing Oligos and Elongation

Nishimura, Onoda

Thanatin fragment (Mini-prep product)

ReagentVolume
TA - F - primer 10 μM1 μL
TA - R - primer 10 μM1 μL
KAPA Taq25 μL
DW23 μL
Total50 μL

(Tm value ≤ -℃)

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation10 cycle
Cycle 26010 secAnnealing10 cycle
Cycle 372℃3030 secElongation10 cycle
Store4℃HoldStore

PCR

Nishimura, Ono, Onoda, Mimata

NdeⅠ - Thanatin - BamHⅠ

ReagentVolume
NdeⅠ - Thanatin - BamHⅠ1 μL
NdeⅠ - F - primer 10 μM1 μL
BamHⅠ - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃30 secAnnealing / Elongation25 cycle
Store4℃HoldStore

PCR

Nishimura, Ono, Onoda, Mimata

BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BamHⅠ - Thanatin - BglⅡ1 μL
BamHⅠ - Asp - Thanatin - R - Neo 10 μM1 μL
BglⅡ - D - Tanatin - R - Neo 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃30 secAnnealing / Elongation25 cycle
Store4℃HoldStore

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 ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

PCR

Ito, Ono, Onoda

Thanatin fragment (derived from annealing TA cloning)

ReagentVolume
Thanatin fragment (derived from annealing TA cloning)1 μL
NdeⅠ - F - primer 10 μM1 μL
BamHⅠ - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation45 cycle
Cycle 265.130 secAnnealing45 cycle
Cycle 368℃30 secElongation45 cycle
Store4℃HoldStore

PCR

Ito, Ono, Onoda

BamHⅠ - Thanatin - BglⅡ

ReagentVolume
Thanatin fragment (derived from annealing TA cloning)1 μL
BamHⅠ - Asp - Thanatin - R - Neo 10 μM1 μL
BglⅡ - D - Tanatin - R - Neo 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation45 cycle
Cycle 266.530 secAnnealing45 cycle
Cycle 368℃30 secElongation45 cycle
Store4℃HoldStore

Digestion

Ono, Onoda

NdeⅠ - Thanatin - BamHⅠ

ReagentVolume
NdeⅠ - Thanatin - BamHⅠ20 μL
NdeⅠ1 μL
BamHⅠ1 μL
10 × K Buffer2 μL
DW5 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
270℃15 minInactivation
Store4℃HoldStore

Digestion

Ono, Onoda

BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BamHⅠ - Thanatin - BglⅡ20 μL
BamHⅠ1 μL
BglⅡ1 μL
10 × K Buffer2 μL
DW5 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
270℃15 minInactivation
Store4℃HoldStore

2015/08/18

Electrophoresis

Nishimura, Ono

Thanatin fragment (PCR 2STEP product)

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 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)

ReagentVolume
TA - F - primer 10 μM5 μL
TA - R - primer 10 μM5 μL
TE 0.8 M NaCl10 μL
Total50 μL

Annealing Oligos and Elongation

StepTemp.TimeProcessCycle
Start94℃2 minInitialization
Step195℃ to 25℃20 minAnnealing
Store4℃HoldStore

PCR

Mimata

Thanatin fragment (derived from annealing)

ReagentVolume
Thanatin fragment (derived from annealing)1 μL
NdeⅠ - F - primer 10 μM1 μL
BamHⅠ - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃30 secAnnealing / Elongation25 cycle
Store4℃HoldStore

PCR

Mimata

Thanatin fragment (derived from annealing)

ReagentVolume
Thanatin fragment (derived from annealing)1 μL
BamHⅠ - Asp - Thanatin - R - Neo 10 μM1 μL
BglⅡ - D - Tanatin - R - Neo 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation25 cycle
Cycle 268℃30 secAnnealing / Elongation25 cycle
Store4℃HoldStore

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

ReagentVolume
NdeⅠ - Thanatin - BamHⅠ20 μL
NdeⅠ1 μL
BamHⅠ1 μL
10 × K Buffer2 μL
DW5 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
270℃15 minInactivation
Store4℃HoldStore

Digestion

Ono, Onoda, Nishimura

BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BamHⅠ - Thanatin - BglⅡ20 μL
BamHⅠ1 μL
BglⅡ1 μL
10 × K Buffer2 μL
DW5 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
270℃15 minInactivation
Store4℃HoldStore

Ligation

Onoda, Mimata

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

ReagentVolume
pGEM T - vector1 μL
Thanatin fragment3 μL
2 × Ligation Buffer5 μL
T4 DNA Ligase1 μL
Total10 μL

Ligation

StepTemp.TimeProcess
14℃6 hourLigation
265℃10 minInactivation
Store4℃HoldStore

2015/08/19

PCR

Ono

Thanatin fragment (derived from annealing)

ReagentVolume
Thanatin fragment (derived from annealing)1 μL
NdeⅠ - F - primer 10 μM1 μL
BamHⅠ - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation40 cycle
Cycle 26030 secAnnealing40 cycle
Cycle 368℃30 secElongation40 cycle
Store4℃HoldStore

PCR

Ono

Thanatin fragment (derived from annealing)

ReagentVolume
Thanatin fragment (derived from annealing)1 μL
BamHⅠ - Thanatin - F 10 μM1 μL
BglⅡ - Tanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 194℃10 secDenaturation40 cycle
Cycle 26030 secAnnealing40 cycle
Cycle 368℃30 secElongation40 cycle
Store4℃HoldStore

Ethanol Precipitation

Mimata, Ono

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

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

Electrophoresis

Mimata

NdeⅠ - Thanatin - BamHⅠ, BamHⅠ - Thanatin - BglⅡ

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

PCR

Ono

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

ReagentVolume
BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ1 μL
BamHⅠ - Thanatin - F 10 μM1 μL
BglⅡ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 268℃30 secAnnealing / Elongation40 cycle
Store4℃HoldStore

PCR

Ono

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 μL
XbaⅠ - Thanatin - F 10 μM1 μL
NdeⅠ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 268℃30 secAnnealing / Elongation40 cycle
Store4℃HoldStore

Ethanol Precipitation

Ono

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

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

Digestion

Ono, Onoda, Mimata, Sakai

NdeⅠ - Thanatin - BamHⅠ

ReagentVolume
NdeⅠ - Thanatin - BamHⅠ20 μL
NdeⅠ1 μL
BamHⅠ1 μL
10 × K Buffer3 μL
DW5 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Digestion

Ono, Onoda, Mimata, Sakai

BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BamHⅠ - Thanatin - BglⅡ20 μL
BamHⅠ1 μL
BglⅡ1 μL
10 × K Buffer3 μL
DW5 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Digestion

Ono, Onoda, Mimata, Sakai

pET - 15b vector, pET - 16b vector

ReagentVolume
pET - 15b vector, pET - 16b vector20 μL
NdeⅠ1 μL
BamHⅠ1 μL
10 × K Buffer3 μL
DW5 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Transformation

Onoda

Thanatin fragment on pGEM - T vector

  1. Added 1 μL of Thanatin fragment on pGEM - T vector to 50 μL of thawed competent cells (DH5α) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Spread 300 μL of the culture onto plate with LBA.
  5. Incubated the plate at 37℃ for 18 hours.

2015/08/20

Electrophoresis

Ono, Nishimura, Mimata

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Electrophoresis

Ono, Nishimura, Mimata

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

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 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)

ReagentVolume
BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ (PCR 2STEP poduct)9 μL
BamHⅠ5 μL
BglⅡ5 μL
10 × K Buffer10 μL
DW71 μL
Total100 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
270℃15 minInactivation
Store4℃HoldStore

Digestion

Ono, Nisimura, Ito

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ (PCR 2STEP poduct)9 μL
XbaⅠ5 μL
SpeⅠ5 μL
10 × K Buffer10 μL
DW71 μL
Total100 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

Digestion

Ono, Nishimura, Ito

BBa_B0015 on pSB1C3

ReagentVolume
BBa_B0015 on pSB1C320 μL
SpeⅠ1 μL
CutSmart Buffer3 μL
DW6 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

Ethanol Precipitation

Ito

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

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

Ito

Electrophoresis

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

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

2015/08/21

PCR

Ono, Nishimura, Onoda

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

ReagentVolume
BamHⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - BglⅡ1 μL
BamHⅠ - Thanatin - F 10 μM1 μL
BglⅡ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 268℃30 secAnnealing / Elongation40 cycle
Store4℃HoldStore

PCR

Ono, Nishimura, Onoda

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 μL
XbaⅠ - Thanatin - F 10 μM1 μL
SpeⅠ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 268℃30 secAnnealing / Elongation40 cycle
Store4℃HoldStore

Electrophoresis

Ono, Onoda, Nishimura

XbaⅠ - Thanatin - SpeⅠ, BamHⅠ- Thanatin - BglⅡ

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Ethanol Precipitation

Ono, Nishimura

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

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

Electrophoresis

Ono, Nishimura

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Ligation

Ono, Nishimura, Ito

BBa_K759012 on pSB1C3 / BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BBa_K759012 on pSB1C35 μL
BamHⅠ - Thanatin - BglⅡ1 μL
10 × T4 DNA Ligase Buffer7 μL
T4 DNA Ligase1 μL
Total14 μL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Ligation

Ono, Nishimura, Ito

BBa_B0015 on pSB1C3 / XbaⅠ - Thanatin - SpeⅠ

ReagentVolume
BBa_B0015 on pSB1C35 μL
XbaⅠ - Thanatin - SpeⅠ1 μL
10 × T4 DNA Ligase Buffer7 μL
T4 DNA Ligase1 μL
Total14 μL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Transformation

Onoda

Thanatin - BBa_K759012 on pSB1C3, Thanatin - BBa_B0015 on pSB1C3

  1. Added 5 μL of Thanatin - BBa_K759012 on pSB1C3, Thanatin - BBa_B0015 on pSB1C3 to 50 μL of thawed competent cells (DH5α) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 200 μL of LB.
  5. Incubated the cells for 2 hrs at 37℃.
  6. Spread 300 μL of the culture onto plate with LBC.
  7. Incubated the plate at 37℃ for 16 hours.

Digestion

Onoda, Ono

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

ReagentVolume
BBa_B0033 on pSB1C3, XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ, XbaⅠ - BBa_K16800020 μL
XbaⅠ1 μL
SpeⅠ1 μL
10 × M Buffer3 μL
DW5 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
280℃20 minInactivation
Store4℃HoldStore

Digestion

Onoda

BBa_E0040 on pSB1C3, BBa_R0040 on pSB1C3

ReagentVolume
BBa_E0040 on pSB1C3, BBa_R0040 on pSB1C320 μL
XbaⅠ1 μL
CutSmart Buffer3 μL
DW6 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
260℃15 minInactivation
Store4℃HoldStore

2015/08/22

Electrophoresis

Ono, Nishimura

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Digestion

Ono, Onoda

BBa_R0010 - BBa_B0034 on pSB1C3

ReagentVolume
BBa_R0010 - BBa_B0034 on pSB1C320 μL
SpeⅠ - HF1 μL
10 × M Buffer5 μL
DW24 μL
Total50 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
Store4℃HoldStore

Electrophoresis

Ono, Onoda

BBa_R0010 - BBa_B0034 on pSB1C3 (Digestion product)

Gel ConcentrationVoltageTimeBuffer
1%100 V45 min1/2 x TBE

Ethanol Precipitation

Ono

BBa_R0010 - BBa_B0034 on pSB1C3 (Digestion product)

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

Colony PCR

Ono, Onoda

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

ReagentVolume
Single Colony-
AGSP - BamHⅠ - Spidroin 10 μM0.4 μL
BglⅡ -D - Thanatin 10 μM0.4 μL
KAPA Taq5 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃30 secDenaturation40 cycle
Cycle 26540 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Ono, Onoda

BBa_B0031 on pSB1A2 (as Positive Control)

ReagentVolume
Single Colony-
100UP - EX - F 10 μM0.4 μL
200DN - PS - R 10 μM0.4 μL
KAPA Taq5 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Electrophoresis

Ono, Onoda

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

Gel ConcentrationVoltageTimeBuffer
1%100 V45 min1/2 x TBE

Ligation

Ono

BBa_R0010 - BBa_B0034 on pSB1C3 / XbaⅠ - Thanatin - SpeⅠ

ReagentVolume
BBa_R0010 - BBa_B0034 on pSB1C35 μL
XbaⅠ - Thanatin - SpeⅠ4 μL
Mighty Mix10 μL
DW1 μL
Total20 μL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Transformation

Ono

BBa_R0010 - BBa_B0034 on pSB1C3

  1. Added 1 μL of BBa_R0010 - BBa_B0034 on pSB1C3 to 50 μL of thawed competent cells (DH5α) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 200 μL of LB.
  5. Incubated the cells for 2 hrs at 37℃.
  6. Spread 300 μL of the culture onto plate with LBC.
  7. Incubated the plate at 37℃ for 16 hours.

PCR

Onoda

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 μL
XbaI - B0034 - XS scar - F 10 μM1 μL
SpeⅠ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

Electrophoresis

Onoda

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

Gel ConcentrationVoltageTimeBuffer
2%50 V50 min1/2 x TBE

PCR

Onoda

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 μL
XbaI - B0034 - XS scar - F 10 μM1 μL
SpeⅠ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

2015/08/23

Electrophoresis

Ono

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 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)

ReagentVolume
Single Colony-
100UP - EX - F 10 μM0.4 μL
SpeⅠ - Thanatin - R 10 μM0.4 μL
KAPA Taq5 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Ito, Ono, Onoda

BBa_B0030 on pSB1A2 (as a positive control)

ReagentVolume
Single Colony-
100UP - EX - F 10 μM0.4 μL
200DN - PS - R 10 μM0.4 μL
KAPA Taq5 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

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)

ReagentVolume
Single Colony-
100UP - EX - F 10 μM0.4 μL
SpeⅠ - Thanatin - R 10 μM0.4 μL
KAPA Taq5 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 25730 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3

ReagentVolume
Single Colony-
AGSP - BamHⅠ - Spidroin 10 μM0.4 μL
BBa_K759012 - R 10 μM0.4 μL
KAPA Taq5 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃30 secDenaturation40 cycle
Cycle 26540 secAnnealing40 cycle
Cycle 372℃40 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Electrophoresis

Ono, Nishimura

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Digestion

Ono, Nishimura

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3

ReagentVolume
BBa_R0010 - BBa_B0034 - Thanatin on pSB1C320 μL
SpeⅠ - HF1 μL
CutSmart Buffer3 μL
DW6 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

Electrophoresis

Ono, Nishimura

BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (digestion product)

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Liquid Culture

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3 into DH5α

ReagentVolume
Single Colony-
LB2000 μL
Chloramphenicol2 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

ReagentVolume
Single Colony-
LB2000 μL
Chloramphenicol2 μL

Cultured for 16 hours.

Sequencing

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3

ReagentVolume
Thanatin - BBa_K759012 on pSB1C31 μL
pbad - f2 / 200 βdomain BBa_K759012 - R1.5 μL
BigDye Terminator1 μL
5 x Sequencing Buffer1.5 μL
DW5 μL
Total10 μL

Sequencing

StepTemp.TimeProcessCycle
Start96℃10 secDenaturation
Cycle 150℃5 sec-30 cycle
Cycle 260℃240 sec-30 cycle
Store4℃HoldStore

Ethanol Precipitation

Ono, Nishimura

Thanatin - BBa_K759012 on pSB1C3

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

PCR

Ono, Nishimura

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 μL
XbaⅠ - BBa_B0034 - XS scar - F 10 μM1 μL
SpeⅠ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 25510 secAnnealing40 cycle
Cycle 368℃30 secElongation40 cycle
Store4℃HoldStore

Ethanol Precipitation

Fujita, Nishimura

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

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

Electrophoresis

Fujita

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

2015/08/26

PCR

Ono、Nishimura

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 μL
XbaⅠ - BBa_B0034 - XS scar - F 10 μM1 μL
SpeⅠ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 25510 secAnnealing40 cycle
Cycle 368℃30 secElongation40 cycle
Store4℃HoldStore

PCR

Ono, Nishimura

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 μL
XbaⅠ - BBa_B0034 - XS scar - F 10 μM1 μL
SpeⅠ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 25330 secAnnealing40 cycle
Cycle 368℃30 secElongation40 cycle
Store4℃HoldStore

PCR

Ono, Nishimura, Fujita

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 μL
XbaⅠ - BBa_B0034 - XS scar - F 10 μM1 μL
SpeⅠ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

Electrophoresis

Ono, Nishimura, Fujita

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Ethanol Precipitation

Fujita, Nishimura, Ono

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

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

Mini-prep

Nishimura, Ono

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

Sequencing

Fujita, Nishimura, Ono

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

ReagentVolume
BBa_R0010 - BBa_B0034 - Thanatin on pSB1C3 (Mini-prep producrt)1 μL
100UP - EX - F / 200DN - PS - R1.5 μL
Ready Reaction Premix1 μL
5 x Sequencing Buffer1.5 μL
DW5 μL
Total10 μL

Sequencing

StepTemp.TimeProcessCycle
Start96℃10 secDenaturation
Cycle 150℃5 sec-30 cycle
Cycle 260℃240 sec-30 cycle
Store4℃HoldStore

Ethanol Precipitation

Fujita, Nishimura, Ono

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

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

2015/08/27

Ligation

Ono, Ito

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

ReagentVolume
BBa_K759012 on pSB1C35 μL
BamHⅠ - Thanatin - BglⅡ4 μL
Mighty Mix10 μL
DW1 μL
Total20 μL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Ligation

Ono, Ito

BBa_K759012 on pSB1C3 (not phosphorylated)

ReagentVolume
BBa_K759012 on pSB1C32 μL
Mighty Mix2 μL
DW6 μL
Total10 μL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Ligation

Ono, Ito

BBa_K759012 on pSB1C3 (phosphorylated)

ReagentVolume
BBa_K759012 on pSB1C32 μL
Mighty Mix2 μL
DW6 μL
Total10 μL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Transformation

Ono, Ito

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

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

Colony PCR

Ito, Ono

Thanatin - BBa_K759012 on pSB1C3

ReagentVolume
Single Colony-
Agsp - BamHⅠ - Spidroin 10 μM0.4 μL
BBa_K759012 - bunit - R / BglⅡ - D - Thanatin - R 10 μM0.4 μL
KAPA Taq5 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation40 cycle
Cycle 26530 secAnnealing40 cycle
Cycle 372℃60 secElongation40 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

2015/08/28

Electrophoresis

Fujita, Ono

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Ethanol Precipitation

Fujita

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

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

Electrophoresis

Fujita, Ono

BamHⅠ - Thanatin - BglⅡ

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 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

ReagentVolume
XbaⅠ - Thanatin - SpeⅠ1 μL
XbaⅠ - Thanatin - F 10 μM1 μL
SpeⅠ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

PCR

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 μL
XbaⅠ - Thanatin - F 10 μM1 μL
SpeⅠ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 26730 secAnnealing30 cycle
Cycle 368℃30 secElongation30 cycle
Store4℃HoldStore

PCR

Fujita, Ono

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 μL
XbaⅠ - Thanatin - F 10 μM1 μL
SpeⅠ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

PCR

Fujita, Ono

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 μL
XbaⅠ - Thanatin - F 10 μM1 μL
SpeⅠ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 26730 secAnnealing30 cycle
Cycle 368℃30 secElongation30 cycle
Store4℃HoldStore

PCR

Fujita, Ono

BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BamHⅠ - Thanatin - BglⅡ1 μL
BamHⅠ - Thanatin - F 10 μM1 μL
BglⅡ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW35 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

PCR

Fujita, Ono

BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BamHⅠ - Thanatin - BglⅡ1 μL
BamHⅠ - Thanatin - F 10 μM1 μL
BglⅡ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 26730 secAnnealing30 cycle
Cycle 368℃30 secElongation30 cycle
Store4℃HoldStore

Electrophoresis

Fujita, Ono

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Digestion

Ono, Fujita

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ20 μL
SpeⅠ1 μL
XbaⅠ1 μL
10 × M Buffer3 μL
0.1%BSA3 μL
DW2 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
Store4℃HoldStore

Digestion

Ono, Fujita

BamHⅠ - Thanatin - BglⅡ

ReagentVolume
BamHⅠ - Thanatin - BglⅡ20 μL
BamHⅠ1 μL
BglⅡ1 μL
10 × K Buffer3 μL
DW5 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
Store4℃HoldStore

2015/08/29

Electrophoresis

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 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

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

Electrophoresis

Fujita, Ono

XbaⅠ - Thanatin - SpeⅠ

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

PCR

Ono

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

ReagentVolume
XbaⅠ - BBa_B0034 - SpeⅠ / XbaⅠ scar - Met - 10 × His Tag - Thanatin - SpeⅠ1 μL
XbaⅠ - Thanatin - F 10 μM1 μL
SpeⅠ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation40 cycle
Cycle 268℃30 secAnnealing / Elongation40 cycle
Store4℃HoldStore

Ethanol Precipitation

Ono

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

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

Electrophoresis

Fujita, Mimata

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

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

Electrophoresis

Fujita, Mimata

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Digestion

Fujita

BBa_R0010 - BBa_B0034 on pSB1C3

ReagentVolume
BBa_R0010 - BBa_B0034 on pSB1C320 μL
SpeⅠ - HF1 μL
CutSmart Buffer3 μL
DW6 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

2015/08/30

Electrophoresis

Mimata

BBa_R0010 - BBa_B0034 on pSB1C3, Thanatin - BBa_K759012 on pSB1C3

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

Digestion

Mimata, Toyooka

XbaⅠ - Thanatin - SpeⅠ

ReagentVolume
XbaⅠ - Thanatin - SpeⅠ20 μL
SpeⅠ1 μL
XbaⅠ1 μL
10 × M Buffer 3 μL
DW5 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
270℃15 minInactivation
Store4℃HoldStore

Digestion

Mimata, Toyooka

BBa_R0040 on pSB1A2, BBa_E0040 on pSB1A2

ReagentVolume
BBa_R0040 on pSB1A2, BBa_E0040 on pSB1A220 μL
SpeⅠ1 μL
CutSmart Buffer3 μL
DW6 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
270℃15 minInactivation
Store4℃HoldStore

Digestion

Mimata, Toyooka

BBa_B0030 on pSB1C3

ReagentVolume
BBa_B0030 on pSB1C320 μL
SpeⅠ1 μL
XbaⅠ1 μL
10 × M Buffer 3 μL
DW5 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
270℃15 minInactivation
Store4℃HoldStore

2015/08/31

PCR

Nishimura, Ono, Toyooka, Fujita

XbaⅠ - Thanatin - SpeⅠ

ReagentVolume
XbaⅠ - Thanatin - SpeⅠ1 μL
XbaⅠ - Thanatin - F 10 μM1 μL
SpeⅠ - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

PCR

Nishimura, Ono, Toyooka, Fujita

BamHⅠ- Thanatin - BglⅡ

ReagentVolume
BamHⅠ- Thanatin - BglⅡ1 μL
BamHⅠ- Thanatin - F 10 μM1 μL
BglⅡ - D - Thanatin - R 10 μM1 μL
KOD - Plus - Neo1 μL
10 x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

Electrophoresis

Ono, Nishimura, Toyooka, Fujita

BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Ethanol Precipitation

Ono, Nishimura, Toyooka, Fujita

BamHⅠ- Thanatin - BglⅡ, XbaⅠ - Thanatin - SpeⅠ

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

Digestion

Ono, Fujita, Toyooka, Nishimura

Thanatin fragment (Ethanol Precipitation product)

ReagentVolume
Thanatin fragment (Ethanol Precipitation product)20 μL
SpeⅠ2 μL
XbaⅠ1 μL
CutSmart Buffer3 μL
DW4 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
Store4℃HoldStore

Digestion

Ono, Fujita, Toyooka, Nishimura

Thanatin fragment (Ethanol Precipitation product)

ReagentVolume
Thanatin fragment (Ethanol Precipitation product)20 μL
BamHⅠ2 μL
BglⅡ6 μL
10 × K Buffer10 μL
DW60 μL
Total100 μL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
Store4℃HoldStore

Ethanol Precipitation

Ono, Nishimura, Toyooka, Fujita

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

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

Electrophoresis

Ono, Nishimura, Toyooka, Fujita, Mimata

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

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 x TBE

Ligation

Ono

BBa_K759012 on pSB1C3 / BamHⅠ- Thanatin - BglⅡ

ReagentVolume
BBa_K759012 on pSB1C395 μL
BamHⅠ- Thanatin - BglⅡ0.5 μL
Mighty Mix10 μL
Total20 μL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
Store4℃HoldStore

Electrophoresis

Ono, Mimata

BBa_K759012 on pSB1C3, BBa_R0010 on pSB1C3

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

Digestion

Onoda,

BBa_E1010 on pSB1C3

ReagentVolume
BBa_E1010 on pSB1C310 μL
EcoRⅠ1 μL
XbaⅠ1 μL
10 × M Buffer 2 μL
DW6 μL
Total20 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Digestion

Onoda

BBa_E1010 on pSB1C3

ReagentVolume
BBa_E1010 on pSB1C310 μL
XbaⅠ1 μL
CutSmart Buffer2 μL
DW7 μL
Total20 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Electrophoresis

Toyooka

BBa_E1010 on pSB1C3 (Digestion product)

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 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

ReagentVolume
HLA, HLZ, BLA, BLZ20 μL
XbaⅠ1 μL
SpeⅠ1 μL
10 × M Buffer 3 μL
DW5 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

Digestion

Ito, Sakai

HLA, HLZ, BLA, BLZ

ReagentVolume
HLA, HLZ, BLA, BLZ10 μL
XbaⅠ1 μL
SpeⅠ1 μL
10 x M buffer 2 μL
DW6 μL
Total20 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

Electrophoresis

Ito, Sakai

HLA family XbaⅠ & SpeⅠ (Digestion product)

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 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

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