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Team:KIT-Kyoto/ぷろとこる英語
From 2011.igem.org
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| - | ・コンピ | + | ・コンピ<br> |
↓Streak E.coli on LB plate and incubate for 16h.<br> | ↓Streak E.coli on LB plate and incubate for 16h.<br> | ||
↓Pick a single colony up, transfer it into 250 ml of SOB medium in a flask. Incubate it at | ↓Pick a single colony up, transfer it into 250 ml of SOB medium in a flask. Incubate it at | ||
18°C with shaking.<br> | 18°C with shaking.<br> | ||
↓When it reaches an OD600 of 0.4 to 0.8, transfer it on an ice for 10 min.<br> | ↓When it reaches an OD600 of 0.4 to 0.8, transfer it on an ice for 10 min.<br> | ||
| - | ↓Harvest cells by centrifugation(4.5 x 10 g) for 10min at 4°C.<br> | + | ↓Harvest cells by centrifugation(4.5 x 10<sup>3</sup> g) for 10min at 4°C.<br> |
↓Remove the supernatant and resuspend the cells in 84 ml of ice-cold TB.<br> | ↓Remove the supernatant and resuspend the cells in 84 ml of ice-cold TB.<br> | ||
↓Keep it on ice for 10min.<br> | ↓Keep it on ice for 10min.<br> | ||
| - | ↓Harvest cells by centrifugation(5 x 10 g) for 10min at 4°C.<br> | + | ↓Harvest cells by centrifugation(5 x 10<sup>3</sup> g) for 10min at 4°C.<br> |
↓Remove the supernatant and resuspend the cells in 40 ml of ice-cold TB.<br> | ↓Remove the supernatant and resuspend the cells in 40 ml of ice-cold TB.<br> | ||
↓Add 1.5 ml of DMSO(7%) and keep it on ice for 10min.<br> | ↓Add 1.5 ml of DMSO(7%) and keep it on ice for 10min.<br> | ||
↓Pour each of 220 µl into chilled eppendorf tubes. Make sure to close tightly and then flash | ↓Pour each of 220 µl into chilled eppendorf tubes. Make sure to close tightly and then flash | ||
freeze in liquid nitrogen and store at -80°C.<br> | freeze in liquid nitrogen and store at -80°C.<br> | ||
Revision as of 09:08, 3 October 2011
いんぐりっしゅ！
Ligation
Refer to following table, prepare a reaction solution.
| insert | 0.5 µl |
| vector | 0.5 µl |
| 2 x Buffer | 2.5 µl |
| T4 ligase | 0.5 µl |
| H2O | 1.0 µl |
| total 5 µl |
Incubate it for 30min at 16 ℃.
・PCR
Add all reagents in a PCR tube.
Based on primers, set an appropriate annealing temperature.
・アガロース電気泳動
↓Prepare a 1% (w/v) agarose gel.
↓Add 10 µl of 6x loading dye to each 50 µl of digested solutions.
↓Load each of 60 µL samples and DNA maker in wells.
↓Run at 50 V and 60min.
↓Stain in EtBr solution for 10min.
・コンピ
↓Streak E.coli on LB plate and incubate for 16h.
↓Pick a single colony up, transfer it into 250 ml of SOB medium in a flask. Incubate it at
18°C with shaking.
↓When it reaches an OD600 of 0.4 to 0.8, transfer it on an ice for 10 min.
↓Harvest cells by centrifugation(4.5 x 103 g) for 10min at 4°C.
↓Remove the supernatant and resuspend the cells in 84 ml of ice-cold TB.
↓Keep it on ice for 10min.
↓Harvest cells by centrifugation(5 x 103 g) for 10min at 4°C.
↓Remove the supernatant and resuspend the cells in 40 ml of ice-cold TB.
↓Add 1.5 ml of DMSO(7%) and keep it on ice for 10min.
↓Pour each of 220 µl into chilled eppendorf tubes. Make sure to close tightly and then flash
