Team:HokkaidoU Japan/Notebook/ecoli

E. coli

March

2015/03/10

Competent Cells

Tanaka

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

実験者

BBa_R0011

ReagentVolume
BBa_R00111 μL
100bpUP-EX-F 10 μM1.5 μL
200bpDN-PS-R 10 μM1.5 μL
KOD Plus NEO1 μL
KOD Plus NEO 10x Buffer5 μL
2 mM dNTPS5 μL
25 mM MgSO43 μL
DW32 μL
Total50 μL

BBa_0030 - BBa_E1010

ReagentVolume
BBa_0030 - BBa_E10101 μL
100bpUP-EX-F 10 μM1.5 μL
200bpDN-PS-R 10 μM1.5 μL
KOD Plus NEO1 μL
KOD Plus NEO 10x Buffer5 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

実験者

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

Digestion

実験者

BBa_R0011

ReagentVolume
BBa_R001144 μL
XbaI1 μL
Cut Smart5 μL
Total50 μL

BBa_0030 - BBa_E1010

ReagentVolume
BBa_0030 - BBa_E101044 μL
XbaI1 μL
Cut Smart5 μL
Total50 μL

Digestion

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

Electrophoresis

実験者

BBa_R0011, BBa_0030 - BBa_E1010

Gel ConcentrationVoltageTimeBuffer
1%100 V30 min2x TBE

Gel Extract

実験者

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

Electrophoresis

実験者

BBa_R0011, BBa_0030 - BBa_E1010

Gel ConcentrationVoltageTimeBuffer
1%100 V30 min2x 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

pET16b

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

Competent Cells

Onoda

rosetta

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

2015/05/27

Transformation

Mimata, Onoda

GFP

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

Transformation

Mimata, Onoda, Ono

mRFP

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

2015/05/29

Mini-prep

Mimata, Onoda, Ono

GFP, mRFP
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

PCR

Onoda, Ono

GFP

ReagentVolume
GFP1 μL
67-F-primer 10 μL1 μL
14-R-primer 10 μL1 μL
KOD FX NEO1 μL
KOD FX NEO 10x Buffer5 μL
2 mM dNTPS5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

mRFP

ReagentVolume
mRFP1 μL
67-F-primer 10 μL1 μL
14-R-primer 10 μL1 μL
KOD FX NEO1 μL
KOD FX 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 cycles
Cycle 268℃60 secAnnealing / Elongation30 cycles
Store4℃HoldStore

2015/05/30

Electrophoresis

Mimata, Onoda, Ono

GFP, mRFP

Gel ConcentrationVoltageTimeBuffer
2%100 A30 min2x TBE

Gel Extract

Onoda, Ono

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

Digestion

Onoda, Ono

GFP

ReagentVolume
GFP20 μL
DW5 μL
Spe11 μL
EcoR11 μL
Cut Smart3 μL
Total30 μL

mRFP

ReagentVolume
mRFP20 μL
DW5 μL
Spe11 μL
EcoR11 μL
Cut Smart3 μL
Total30 μL

Digestion

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

Digestion

Onoda, Ono

pET15b

ReagentVolume
pET15b10 μL
DW6 μL
Spe11 μL
EcoR11 μL
Cut Smart2 μL
Total20 μL

pET16b

ReagentVolume
pET16b10 μL
DW6 μL
Spe11 μL
EcoR11 μL
Cut Smart2 μL
Total20 μL

pSB1A3

ReagentVolume
pSB1A310 μL
DW6 μL
Spe11 μL
EcoR11 μL
Cut Smart2 μL
Total20 μL

pSB4C5

ReagentVolume
pSB4C52 μL
DW14 μL
Spe11 μL
EcoR11 μL
Cut Smart2 μL
Total20 μL

Digestion

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

2015/05/31

Electrophoresis

Mimata, Onoda, Ono

GFP, RFP, pET15b, pET16b, pSB1A3, pSB4C5

Gel ConcentrationVoltageTimeBuffer
1%100 V30 min1/2x TBE

Gel Extract

Mimata, Onoda, Ono

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

Electrophoresis

Mimata, Onoda, Ono

GFP, mRFP

Gel ConcentrationVoltageTimeBuffer
1%100 V30 min1/2x TBE

PCR

Mimata, Onoda

GFP

ReagentVolume
GFP1 μL
67-F-primer 10 μM1 μL
14-R-primer 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

mRFP

ReagentVolume
mRFP1 μL
67-F-primer 10 μM1 μL
14-R-primer 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

GFP

ReagentVolume
GFP16 μL
Spe11 μL
EcoR11 μL
Cut Smart2 μL
Total20 μL

mRFP

ReagentVolume
mRFP16 μL
Spe11 μL
EcoR11 μL
Cut Smart2 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 forward primer1 μL
TA reverse 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 A30 min2x TBE
→failed

Annealing Oligos and Elongation

実験者

thanatin fragment for TA cloning

ReagentVolume
TA-forward primer 1 μM1 μL
TA-reverse 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

実験者

thanatin fragment for TA cloning

Gel ConcentrationVoltageTimeBuffer
2%100V30min2x TBE

Gel Extract

実験者

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

Ligation

実験者

thanatin fragment for TA cloning / Tvector pGEM

ReagentVolume
Tvector pGEM1.7μL
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

実験者

Tvector pGEM

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

July

2015/07/25

Transformation

Onoda

dT on pSB1C3

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

Transformation

Onoda

pLac - B0034 on pSB1C3

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

Transformation

Onoda

pTet - B0034 on pSB1C3

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

Transformation

Onoda

B0034 on pSB1A2

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

PCR

Onoda

dT on pSB1C3

ReagentVolume
10% dT on pSB1C31 μL
100bpUP-EX-F 10 μM1 μL
200bpDN-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

pLac - B0034 on pSB1C3

ReagentVolume
10% pLac - B0034 on pSB1C31 μL
100bpUP-EX-F 10 μM1 μL
200bpDN-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

pTet - B0034 on pSB1C3

ReagentVolume
10% pTet - B0034 on pSB1C31 μL
100bpUP-EX-F 10 μM1 μL
200bpDN-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

B0034 on pSB1A2

ReagentVolume
10% B0034 on pSB1A21 μL
100bpUP-EX-F 10 μM1 μL
200bpDN-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

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

dT on pSB1C3, pLac - B0034 on pSB1C3, pTet - B0034 on pSB1C3, B0034 on pSB1A2

Gel ConcentrationVoltageTimeBuffer
2%100 A30 min2x TBE

2015/07/26

Liquid Culture

Ono

pTet - B0034 on pSB1A2

ReagentVolume
Single Colony-
LB2000 μL
Ampicillin2 μL

Cultured for 15 hours.

Mini-prep

実験者

pLac - B0034 on pSB1C3, dT on pSB1C3, B0034 on pSB1A2
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Electrophoresis

実験者

pTet - B0034 on pSB1C3, pLac - B0034 on pSB1C3, B0034 on pSB1A3, dT on pSB1C3

Gel ConcentrationVoltageTimeBuffer
2%100 A30 min2x TBE

Gel Extract

Ono

pLac - B0034 on pSB1C3, pTet - B0034 on pSB1C3, B0034 on pSB1A2, dT on pSB1C3
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel

2015/07/27

Mini-prep

Ono

pTet - B0034 on pSB1A2
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

August

September

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