Team:HokkaidoU Japan/Notebook/ecoli
E. coli
March
2015/03/10
Competent Cells
mami
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
実験者
BBa_R0011
Reagent | Volume |
---|---|
BBa_R0011 | 1 μL |
100bpUP-EX-F 10 μM | 1.5 μL |
200bpDN-PS-R 10 μM | 1.5 μL |
KOD Plus NEO | 1 μL |
KOD Plus NEO 10x Buffer | 5 μL |
2 mM dNTPS | 5 μL |
25 mM MgSO4 | 3 μL |
DW | 32 μL |
Total | 50 μL |
BBa_0030 - BBa_E1010
Reagent | Volume |
---|---|
BBa_0030 - BBa_E1010 | 1 μL |
100bpUP-EX-F 10 μM | 1.5 μL |
200bpDN-PS-R 10 μM | 1.5 μL |
KOD Plus NEO | 1 μL |
KOD Plus NEO 10x Buffer | 5 μL |
2 mM dNTPS | 5 μL |
25 mM MgSO4 | 3 μL |
DW | 32 μL |
Total | 50 μL |
3 Step Cycle (Tm value ≤ 63℃)
Step | Temp. | Time | Process | Cycle |
---|---|---|---|---|
Start | 94℃ | 120 sec | Initialization | |
Cycle 1 | 98℃ | 10 sec | Denaturation | 30 cycle |
Cycle 2 | 62.6℃ | 30 sec | Annealing | 30 cycle |
Cycle 3 | 68℃ | 1 min | Elongation | 30 cycle |
Store | 4℃ | Hold | Store |
PCR Purification
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
Reagent | Volume |
---|---|
BBa_R0011 | 44 μL |
XbaI | 1 μL |
Cut Smart | 5 μL |
Total | 50 μL |
BBa_0030 - BBa_E1010
Reagent | Volume |
---|---|
BBa_0030 - BBa_E1010 | 44 μL |
XbaI | 1 μL |
Cut Smart | 5 μL |
Total | 50 μL |
Digestion
Step | Temp. | Time | Process |
---|---|---|---|
1 | 37℃ | 300 min | Digestion |
2 | 60℃ | 15 min | Inactivation |
Store | 4℃ | Hold | Store |
Electrophoresis
実験者
BBa_R0011, BBa_0030 - BBa_E1010
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
1% | 100 V | 30 min | 2x 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 Concentration | Voltage | Time | Buffer |
---|---|---|---|
1% | 100 V | 30 min | 2x TBE |
May
2015/05/13
Transformation
Onoda
pET15b
- Added 1 μL of pET15b to 50 μL of thawed competent cells (DH5alpha) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Spread 50 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 16 hours.
Transformation
Onoda
pET16b
- Added 1 μL of pET16b to 50 μL of thawed competent cells (DH5alpha) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Spread 50 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 16 hours.
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 培養時間 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
GFP
- Added 1 μL of GFP to thawed competent cells (Rosetta and DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Spread 300 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 16 hours.
Transformation
Mimata, Onoda, Ono
mRFP
- Added 1 μL of mRFP to thawed competent cells (Rosetta and DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Spread 300 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 16 hours.
2015/05/29
Mini-prep
Mimata, Onoda, Ono
GFP, mRFP
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol
PCR
Onoda, Ono
GFP
Reagent | Volume |
---|---|
GFP | 1 μL |
67-F-primer 10 μL | 1 μL |
14-R-primer 10 μL | 1 μL |
KOD FX NEO | 1 μL |
KOD FX NEO 10x Buffer | 5 μL |
2 mM dNTPS | 5 μL |
25 mM MgSO4 | 3 μL |
DW | 33 μL |
Total | 50 μL |
mRFP
Reagent | Volume |
---|---|
mRFP | 1 μL |
67-F-primer 10 μL | 1 μL |
14-R-primer 10 μL | 1 μL |
KOD FX NEO | 1 μL |
KOD FX NEO 10x Buffer | 5 μL |
2 mM dNTPS | 5 μL |
25 mM MgSO4 | 3 μL |
DW | 33 μL |
Total | 50 μL |
2 Step Cycle (Tm value ≥ 63℃)
Step | Temp. | Time | Process | Cycle |
---|---|---|---|---|
Start | 94℃ | 120 sec | Initialization | |
Cycle 1 | 98℃ | 10 sec | Denaturation | 30 cycles |
Cycle 2 | 68℃ | 60 sec | Annealing / Elongation | 30 cycles |
Store | 4℃ | Hold | Store |
2015/05/30
Electrophoresis
Mimata, Onoda, Ono
GFP, mRFP
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
2% | 100 A | 30 min | 2x TBE |
Gel Extract
Onoda, Ono
GFP, mRFP
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
Digestion
Onoda, Ono
GFP
Reagent | Volume |
---|---|
GFP | 20 μL |
DW | 5 μL |
Spe1 | 1 μL |
EcoR1 | 1 μL |
Cut Smart | 3 μL |
Total | 30 μL |
mRFP
Reagent | Volume |
---|---|
mRFP | 20 μL |
DW | 5 μL |
Spe1 | 1 μL |
EcoR1 | 1 μL |
Cut Smart | 3 μL |
Total | 30 μL |
Digestion
Step | Temp. | Time | Process |
---|---|---|---|
1 | 37℃ | 120 min | Digestion |
2 | 60℃ | 15 min | Inactivation |
Store | 4℃ | Hold | Store |
Digestion
Onoda, Ono
pET15b
Reagent | Volume |
---|---|
pET15b | 10 μL |
DW | 6 μL |
Spe1 | 1 μL |
EcoR1 | 1 μL |
Cut Smart | 2 μL |
Total | 20 μL |
pET16b
Reagent | Volume |
---|---|
pET16b | 10 μL |
DW | 6 μL |
Spe1 | 1 μL |
EcoR1 | 1 μL |
Cut Smart | 2 μL |
Total | 20 μL |
pSB1A3
Reagent | Volume |
---|---|
pSB1A3 | 10 μL |
DW | 6 μL |
Spe1 | 1 μL |
EcoR1 | 1 μL |
Cut Smart | 2 μL |
Total | 20 μL |
pSB4C5
Reagent | Volume |
---|---|
pSB4C5 | 2 μL |
DW | 14 μL |
Spe1 | 1 μL |
EcoR1 | 1 μL |
Cut Smart | 2 μL |
Total | 20 μL |
Digestion
Step | Temp. | Time | Process |
---|---|---|---|
1 | 37℃ | 120 min | Digestion |
2 | 60℃ | 15 min | Inactivation |
Store | 4℃ | Hold | Store |
2015/05/31
Electrophoresis
Mimata, Onoda, Ono
GFP, RFP, pET15b, pET16b, pSB1A3, pSB4C5
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
1% | 100 V | 30 min | 1/2x 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 Concentration | Voltage | Time | Buffer |
---|---|---|---|
1% | 100 V | 30 min | 1/2x TBE |
PCR
Mimata, Onoda
GFP
Reagent | Volume |
---|---|
GFP | 1 μL |
67-F-primer 10 μM | 1 μL |
14-R-primer 10 μM | 1 μL |
KOD Plus NEO | 1 μL |
KOD Plus NEO 10x Buffer | 5 μL |
2 mM dNTPS | 5 μL |
25 mM MgSO4 | 3 μL |
DW | 33 μL |
Total | 50 μL |
mRFP
Reagent | Volume |
---|---|
mRFP | 1 μL |
67-F-primer 10 μM | 1 μL |
14-R-primer 10 μM | 1 μL |
KOD Plus NEO | 1 μL |
KOD Plus NEO 10x Buffer | 5 μL |
2 mM dNTPS | 5 μL |
25 mM MgSO4 | 3 μL |
DW | 33 μL |
Total | 50 μL |
2 Step Cycle (Tm value ≥ 63℃)
Step | Temp. | Time | Process | Cycle |
---|---|---|---|---|
Start | 94℃ | 120 sec | Initialization | |
Cycle 1 | 98℃ | 10 sec | Denaturation | 30 cycle |
Cycle 2 | 68℃ | 60 sec | Annealing / Elongation | 30 cycle |
Store | 4℃ | Hold | Store |
June
2015/06/10
Digestion
Mimata, Onoda
GFP
Reagent | Volume |
---|---|
GFP | 16 μL |
Spe1 | 1 μL |
EcoR1 | 1 μL |
Cut Smart | 2 μL |
Total | 20 μL |
mRFP
Reagent | Volume |
---|---|
mRFP | 16 μL |
Spe1 | 1 μL |
EcoR1 | 1 μL |
Cut Smart | 2 μL |
Total | 20 μL |
Digestion
Step | Temp. | Time | Process |
---|---|---|---|
1 | 37℃ | 600 min | Digestion |
2 | 60℃ | 15 min | Inactivation |
Store | 4℃ | Hold | Store |
2015/06/16
PCR
Mimata
thanatin fragment for TA cloning
Reagent | Volume |
---|---|
TA forward primer | 1 μL |
TA reverse primer | 1 μL |
Kapa Taq | 25 μL |
DW | 23 μL |
Total | 50 μL |
3 Step Cycle (Tm value ≤ 63℃)
Step | Temp. | Time | Process | Cycle |
---|---|---|---|---|
Start | 95℃ | 180 sec | Initialization | |
Cycle 1 | 95℃ | 30 sec | Denaturation | 35 cycle |
Cycle 2 | 63℃ | 30 sec | Annealing | 35 cycle |
Cycle 3 | 72℃ | 10 sec | Elongation | 35 cycle |
72℃ | 60 sec | |||
Store | 4℃ | Hold | Store |
2015/06/17
Electrophoresis
Mimata
thanatin fragment for TA cloning
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
2% | 100 A | 30 min | 2x TBE |
Annealing Oligos and Elongation
実験者
thanatin fragment for TA cloning
Reagent | Volume |
---|---|
TA-forward primer 1 μM | 1 μL |
TA-reverse primer 1 μM | 1 μL |
Kapa Taq | 25 μL |
DW | 23 μL |
Total | 50 μL |
Annealing Oligos and Elongation
Step | Temp. | Time | Process | Cycle |
---|---|---|---|---|
Start | 95℃ | 1 min | Initialization | |
Cycle1 | 95℃ to 23℃ | 45 min | Annealing | 1 cycle |
Cycle 2 | 72℃ | 1 min | Elongation | 1 cycle |
Store | 4℃ | Hold | Store |
2015/06/19
Electrophoresis
実験者
thanatin fragment for TA cloning
Gel Concentration | Voltage | Time | Buffer |
---|---|---|---|
2% | 100V | 30min | 2x TBE |
Gel Extract
実験者
thanatin fragment for TA cloning
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
DNA extraction from gel
Ligation
実験者
thanatin fragment for TA cloning / Tvector pGEM
Reagent | Volume |
---|---|
Tvector pGEM | 1.7μL |
thanatin fragment for TA cloning | 0.15μL |
Mighty Mix | 1.85μL |
T4 Ligase | 0.18μL |
DW | 6.12μL |
Total | 10μL |
Ligation
Step | Temp. | Time | Process |
---|---|---|---|
1 | 16℃ | 30 min | Ligation |
2 | 65℃ | 10 min | Inactivation |
Store | 4℃ | Hold | Store |
2015/06/21
Transformation
実験者
Tvector pGEM
- Added 1 μL of thanatin fragment to 50 μL of thawed competent cells (rosseta/DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Spread 300 μL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 19 hours.