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From 2011.igem.org

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 10 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 10 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

freeze in liquid nitrogen and store at -80°C.