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Team:Hong Kong HKU/TEM
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BL21(DE3) cells carrying the plasmids pETE and pETES-mCherry was inoculated in 10ml of LB and allowed to grow to OD ~0.8 at 37°C. They were then transferred to a 30°C shaking incubator at 200rpm for overnight incubation. The next day 1ml of cells were collected and washed once briskly with 1% sodium cacodylate buffer. The cells were then first fixed with 2.5% glutaraldehyde in 1% sodium cacodylate buffer for 20min, washed three times afterwards, and second fixed with 1% osmium tetroxide in 1% sodium cacodylate buffer for 30min, and washed for another three times. 1% agarose was added to the pre-warmed sample and immediately centrifuged for 2,000rpm for 10min. The set agarose embedment was extracted, cut into 2mm blocks, and dehydr ated with a series of 50%, 70%, 90%, 100% ethanol, finally with propylene oxide. A 1:1 mixture of epoxy resin and propylene oxide was then added to infiltrate the blocks with the resin at 37°C overnight. The next day the blocks were transferred to pure epoxy resin and allowed to polymerize at 65°C. Semi-thin and ultra-thin sections were obtained using an ultratome and collected on water. Sections were stained with uranyl acetate and visualized using Philips CM100 TEM. | BL21(DE3) cells carrying the plasmids pETE and pETES-mCherry was inoculated in 10ml of LB and allowed to grow to OD ~0.8 at 37°C. They were then transferred to a 30°C shaking incubator at 200rpm for overnight incubation. The next day 1ml of cells were collected and washed once briskly with 1% sodium cacodylate buffer. The cells were then first fixed with 2.5% glutaraldehyde in 1% sodium cacodylate buffer for 20min, washed three times afterwards, and second fixed with 1% osmium tetroxide in 1% sodium cacodylate buffer for 30min, and washed for another three times. 1% agarose was added to the pre-warmed sample and immediately centrifuged for 2,000rpm for 10min. The set agarose embedment was extracted, cut into 2mm blocks, and dehydr ated with a series of 50%, 70%, 90%, 100% ethanol, finally with propylene oxide. A 1:1 mixture of epoxy resin and propylene oxide was then added to infiltrate the blocks with the resin at 37°C overnight. The next day the blocks were transferred to pure epoxy resin and allowed to polymerize at 65°C. Semi-thin and ultra-thin sections were obtained using an ultratome and collected on water. Sections were stained with uranyl acetate and visualized using Philips CM100 TEM. | ||
Revision as of 02:50, 18 October 2014
Details
BL21(DE3) cells carrying the plasmids pETE and pETES-mCherry was inoculated in 10ml of LB and allowed to grow to OD ~0.8 at 37°C. They were then transferred to a 30°C shaking incubator at 200rpm for overnight incubation. The next day 1ml of cells were collected and washed once briskly with 1% sodium cacodylate buffer. The cells were then first fixed with 2.5% glutaraldehyde in 1% sodium cacodylate buffer for 20min, washed three times afterwards, and second fixed with 1% osmium tetroxide in 1% sodium cacodylate buffer for 30min, and washed for another three times. 1% agarose was added to the pre-warmed sample and immediately centrifuged for 2,000rpm for 10min. The set agarose embedment was extracted, cut into 2mm blocks, and dehydr ated with a series of 50%, 70%, 90%, 100% ethanol, finally with propylene oxide. A 1:1 mixture of epoxy resin and propylene oxide was then added to infiltrate the blocks with the resin at 37°C overnight. The next day the blocks were transferred to pure epoxy resin and allowed to polymerize at 65°C. Semi-thin and ultra-thin sections were obtained using an ultratome and collected on water. Sections were stained with uranyl acetate and visualized using Philips CM100 TEM.
