Team:Botchan Lab Tokyo/Parts
Botchan Lab. Tokyo
Parts
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.