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Revision as of 13:17, 2 October 2011 (view source) Adachi (Talk | contribs) ← Older edit		Revision as of 09:03, 3 October 2011 (view source) Tooshi (Talk | contribs) Newer edit →
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	<BR>		<BR>
		+
		+	・PCR<br>
		+	Add all reagents in a PCR tube.<br>
		+	Based on primers, set an appropriate annealing temperature.<br>
		+
		+	・アガロース電気泳動<br>
		+	↓Prepare a 1% (w/v) agarose gel.<br>
		+	↓Add 10 µl of 6x loading dye to each 50 µl of digested solutions.<br>
		+	↓Load each of 60 µL samples and DNA maker in wells.<br>
		+	↓Run at 50 V and 60min.<br>
		+	↓Stain in EtBr solution for 10min.<br>
		+
		+
		+	・コンピ
		+	↓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
		+	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>
		+	↓Harvest cells by centrifugation(4.5 x 10 g) for 10min at 4°C.<br>
		+	↓Remove the supernatant and resuspend the cells in 84 ml of ice-cold TB.<br>
		+	↓Keep it on ice for 10min.<br>
		+	↓Harvest cells by centrifugation(5 x 10 g) for 10min at 4°C.<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>
		+	↓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>

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Revision as of 09:03, 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 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.