Team:Botchan Lab Tokyo/Protocols
Botchan Lab. Tokyo
Protocols
1 Genetic modification experiment protocol
1. Making culture medium
TGY broth
| material | concentration(5%) |
|---|---|
| Bacto tryptone | 0.5 |
| Bacto yeast ectract | 0.3 |
| Glucose | 0.1 |
| H2O | - |
| pH | 7.0±2.0 |
TGY agar plate
| material | concentration(5%) |
|---|---|
| Bacto tryptone | 1.0 |
| Bacto yeast ectract | 0.5 |
| Glucose | 1.0 |
| Bact agar | 1.5 |
| H2O | - |
| pH | 7.0±2.0 |
LB broth
| material | concentration(5%) |
|---|---|
| Bacto tryptone | 1.0 |
| Bacto yeast ectract | 0.5 |
| NaCl | 1.0 |
| H2O | - |
| pH | 7.0±2.0 |
LB agar plate
| material | concentration(5%) |
|---|---|
| Bacto tryptone | 1.0 |
| Bacto yeast ectract | 0.5 |
| NaCl | 1.0 |
| agar | 1.5 |
| H2O | - |
| pH | 7.0±2.0 |
All culture medium is autoclaved.
2. Pre-culture (D.radiodurans)
About 5 mL TGY broth dispense for tube. Put D.radiodurans into tube Shaking culture at 30 ℃
3. Culture(D.radiodurans)
Swab Pre-cultured D.radiodurans to TGY agar plate and LB p late using bacteria spreader incubate at 30 ℃ all night
4. Colony PCR
Dispensing reagent for 1.5 mL tube
Dispensing primer to each PCR tube
Transfer a few cells from each colony to a corresponding PCR tube
Perform PCR
After PCR was complete, each reaction is subjected to electrophoresis.
5. Electrophoresis
Mix agarose S and 1×TAE, and make agarose gel
Add 6× loading buffer to each of DNA sample
Load a one step ladder and DNA sample into each well of the gel
Run the gel at 100 v until the dye line is approximately 75-80 % of the
way down the gel.
Place the gel into a container filled with EtBr for 20-30 mins
Using transilluminator, visualize DNA fragments
6. Gel extraction
We use illustraTM GFXTM PCR DNA and Gel Band Purification Kit
Weigh a DNase-free 1.5 mL microcentrifuge tube
Excise band of interest and place in microcentrifuge tube
Weigh microcentrifuge tube plus agarose gel band
Calculate weight of agarose gel slicebr
Add 10 μL Capture buffer type 3 for each 10 mg agarose gel slice
Mix by inversion
Incubate 60°C until agarose is completely dissolved
Check color of Capture buffer type 3-sample mix is yellow or pale orange
Add 600 μL Capture buffer type 3-sample mix to assembled
GFX MicroSpin™ column and Collection tube
Incubate 60 s room temperature
Spin 30 s 16 000 × g. Discard flow through
Place GFX MicroSpin column inside the same Collection tube
Repeat Sample Binding step until all sample is loaded
Add 500 μL Wash buffer type 1
Spin 30 s 16 000 × g
Discard Collection tube. Transfer GFX MicroSpin column to a clean 1.5mL
DNase-free microcentrifuge tube.
Add 10–50 μL Elution buffer type 4 or type 6
Incubate 60 s room temperature
Spin 60 s 16 000 × g
Retain flow through
Store purified sample DNA at -20°C
7. Inverse PCR
Dispensing reagent for PCR tube
Perform PCR
After PCR was complete, each reaction is subjected to electrophoresis.
8. Transformation
Thaw competent cells on ice.
Chill approximately of the ligation mixture in a microcentrifuge tube.
Add competent cells to the DNA.
Place the mixture on ice for 30 minutes. Do not mix.
Heat shock at 42°C for 45 seconds*. Do not mix.
Add SOC culture medium
Transfer the entire amount to aseptic tube
Place tube at 37°C for 60 minutes.
Shake vigorously (10,000 rpm 10 min 4 ℃)
Discard the supernatant
The cells and ligation mixture onto the plates.
Incubate overnight at 37°C
9. Infusion cloning
Mix the reagents
Using Thermo stat plus, incubate the reaction for 15 min at 50 ℃
Store the cloning reactions at -20 ℃ for all night
10. Plasmid extraction
We use QIAprep Spin Miniprep Kit
Spin culture fluid 10 min 15000 rpm
Resuspend pelleted bacterial cells in 250 µL of Buffer P1 and transfer to a 1.5 mL tube.
Add 250 µL of Buffer P2 and gently invert the tube 4-6 times to mix.
Add 350 µL of Buffer N3 and invert the tube immediately but gently 4-6 times
Spin 13000 rpm 10 min 4 ℃
Transfer the supernatant (800 µL) to QIAprep 2.0 spin column.
Spin 13000 rpm 10 min 4 ℃
Discard flow through
Add 0.5 ml PB buffer
Spin 13000 rpm 60 s 4 ℃
Discard flow through
Wash QIAprep spin column by adding 0.75 ml of PE Buffer and centrifuging 60 s
Discard flow through
Centrifuge for an additional 1 min to remove residual wash buffer
Put QIAprep spin column to 1.5 ml tube and add 50 µL buffer EB
Let stand for 1 min
Centrifuge for 1 min
Subjected to electrophoresis
2.Radiation irradiation experiment Protocol
1. Making culture medium
TGY broth
| material | concentration(g/100mL H2O) |
|---|---|
| Bacto tryptone | 0.5 |
| Bacto yeast extract | 0.3 |
| Glucose | 0.1 |
| pH | 7.0±02 |
LB+Cm broth
| material | concentration(g/100mL H2O) |
|---|---|
| Bacto tryptone | 1 |
| Bacto yeast extract | 0.5 |
| NaCl | 1 |
| Chloramphenicol aq | 20 μg/mL |
| pH | 7.0±02 |
LB+Cm plate
| material | concentration(g/100mL H2O) |
|---|---|
| Bacto tryptone | 1 |
| Bacto yeast extract | 0.5 |
| NaCl | 1 |
| Bacto agar | 2 |
| Chloramphenicol aq | 20 μg/mL |
| pH | 7.0±02 |
TGY plate
| material | concentration(g/100mL H2O) |
|---|---|
| Bacto tryptone | 1 |
| Bacto yeast extract | 0.3 |
| Glucose | 0 |
| Bacto aga | 1.5 |
| pH | 7.0±02 |
2.Preparation Butterfield’s Phosphate Buffer
| material | volume |
|---|---|
| potassium dihydorogen phosphate | 34.0g |
| H2o | 1000mL |
| pH | 7.0±0.2 |
3.Pre-culture(Deinococcus.radiodurans)
Prepare master plate of D.radiodurans
Dispense 3 mL* TGY broth to sterile tube
Transfer a few cells from master plate’s colony to the tube
Shaking culture at 37 ℃ over night (18-20 hours)
*This is for one irradiated sample. When the number of irradiated samples were more than one, we added extra 1 mL for an increment.
4.Pre-culture (Escherichia.coli DH5α with empty vector)
Prepare master plate of E.coli with vector
Dispense 3 mL* LB+Cm broth to sterile tube
Transfer a few cells from master plate’s colony to the tube
Shaking culture at 37 ℃ over night (18-20 hours)
*This is for one irradiated sample. When the number of irradiated samples were more than one, we added extra 1 mL for an increment.
5.Preparation of gamma-irradiation samples
Dispense 1 mL pre-cultured bacterial solution into each 1.5 mL tube
Centrifuge 2400×g 10 min 4 ℃
Discard flow through
Add 1 mL Butterfield’s Phosphate Buffer (BPB) and suspend
Wrap the parafilm around the tube and leave it on ice for 30~60 min
6.Gamma irradiation
Measure the temperature in the gamma cell* chamber
Place gamma-irradiation samples in gamma cell
Irradiate a certain amount of gamma ray (10, 50, 100 Gy)
*The gamma cell irradiates 0.714-0.715 Gy/min. So, we irradiated certain range of time to reach the objective doses.
7.Dilution and plating of the cells
Dilute gamma-irradiated samples with BPB solution using tenfold dilution
Plate 100 µL each dilution for plate (Sow E.coli for LB+Cm plate. Sow D.radiodurans for TGY plate) Sow each diluted samples for 2 plates
Wrap the parafilm around the plate and incubate at 30 ℃ for 48-72 hours*
Count the number of colonies
*The incubation was continued until the colonies were large enough to be counted.
Each experiment was conducted independently 3 times.
8. The calculation of survival rate
The average Colony Forming Units (CFU) / mL was calculated from the numbers of colonies on the plate for irradiated groups and a non-irradiated group, respectively (see the number of colonies on lab note). The average CFU / mL of an irradiated sample (N) was divided by the average CFU / mL of a non-irradiated sample (N0) to produce the survival rate (N0/N). As for 0 Gy, an irradiated sample was as same as a non-irradiated group, so the survival rate was calculated as always 1 (N0/N0). Each experiment was conducted independently 3 times. References ・Sommers CH, Rajkowski KT, 2008, Inactivation of Escherichia coli JM109, DH5alpha, and O157:H7 suspended in Butterfield's Phosphate Buffer by gamma irradiation., Journal of food science, 73(2):M87-90.
3. characterization
1. recA
1-1. LB broth(50 mL), LB broth+Cm(100 mL).
We made LB broth(50 mL), LB broth+Cm(100 mL). We added Bacto tryptone 0.5 g, Bacto yeast extract 0.25 g and NaCl 0.5 g and then adjusted the pH to 7±0.2, lastly added distilled water up to 50 mL. We added Bacto tryptone 1 g, Bacto yeast extract 0.5 g, NaCl 1 g and Cm solution(100 mL) 20 µL and then adjusted the pH to 7±0.2, lastly added distilled water up to 100 mL.
1-2. precultured
We dispensed liquid culture medium 10 mL in a culture tube and precultured (37℃).At this time, we used LB broth in preculturing competent cell.
1-3. Dispensed
We dispensed liquid culture medium 10 mL in 6 (3×2) culture tubes and inoculationed bacterial culture collected from culture tubes with preculturing.At this time, we only inoculationed the competent cell in culture tube dispensed LB broth.
1-4. turbidity
We measured turbidity until 300 minutes later by 30 minutes with shaking culture in 37℃. When we measured, we stopped shaking culture, dispensed 1 mL in 1.5 mL tubes with pipette. We kept in mind that measure the turbidity which the moment of inoculationing as the time of 0 minute later.
1-5. growth curve
We made growth curve from turbidity.
2. RFP
Same as 2.Radiation irradiation experiment Protocol above.