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.

Latest revision as of 02:51, 18 October 2014

Transmission Electron Microscopy

Transmission Electron Microscopy

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.