Cell culture
Difference between revisions of "Team:SCUT-China A/Protocols"
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} | } | ||
| − | # | + | #u2271 { |
position:absolute; | position:absolute; | ||
left:150px; | left:150px; | ||
| Line 1,438: | Line 1,436: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
} | } | ||
| − | # | + | #u2271_div.mouseOver { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,467: | Line 1,465: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
} | } | ||
| − | # | + | #u2271.mouseOver { |
} | } | ||
| − | # | + | #u2272 { |
position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 1,476: | Line 1,474: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
} | } | ||
| − | # | + | #u2273_div { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,506: | Line 1,504: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
} | } | ||
| − | # | + | #u2273 { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,518: | Line 1,516: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
} | } | ||
| − | # | + | #u2273_div.mouseOver { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,548: | Line 1,546: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
} | } | ||
| − | # | + | #u2273.mouseOver { |
} | } | ||
| − | # | + | #u2274 { |
position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 1,557: | Line 1,555: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
} | } | ||
| − | # | + | #u2275 { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,564: | Line 1,562: | ||
height:0px; | height:0px; | ||
} | } | ||
| − | # | + | #u2276_div { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,583: | Line 1,581: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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| − | # | + | #u2276 { |
position:absolute; | position:absolute; | ||
left:918px; | left:918px; | ||
| Line 1,595: | Line 1,593: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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| − | # | + | #u2276_div.selected { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,614: | Line 1,612: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
} | } | ||
| − | # | + | #u2276.selected { |
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| − | # | + | #u2277 { |
position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 1,623: | Line 1,621: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
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| − | # | + | #u2278 { |
position:absolute; | position:absolute; | ||
left:717px; | left:717px; | ||
| Line 1,632: | Line 1,630: | ||
visibility:hidden; | visibility:hidden; | ||
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| − | # | + | #u2278_state0 { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,644: | Line 1,642: | ||
background-image:none; | background-image:none; | ||
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| − | # | + | #u2278_state0_content { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,651: | Line 1,649: | ||
height:1px; | height:1px; | ||
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| − | # | + | #u2279_div { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,680: | Line 1,678: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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| − | # | + | #u2279 { |
position:absolute; | position:absolute; | ||
left:450px; | left:450px; | ||
| Line 1,692: | Line 1,690: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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| − | # | + | #u2279_div.mouseOver { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,721: | Line 1,719: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
} | } | ||
| − | # | + | #u2279.mouseOver { |
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| − | # | + | #u2280 { |
position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 1,730: | Line 1,728: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
} | } | ||
| − | # | + | #u2281_div { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,760: | Line 1,758: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
} | } | ||
| − | # | + | #u2281 { |
position:absolute; | position:absolute; | ||
left:300px; | left:300px; | ||
| Line 1,772: | Line 1,770: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
} | } | ||
| − | # | + | #u2281_div.mouseOver { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,802: | Line 1,800: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
} | } | ||
| − | # | + | #u2281.mouseOver { |
} | } | ||
| − | # | + | #u2282 { |
position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 1,811: | Line 1,809: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
} | } | ||
| − | # | + | #u2283_div { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,839: | Line 1,837: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
} | } | ||
| − | # | + | #u2283 { |
position:absolute; | position:absolute; | ||
left:150px; | left:150px; | ||
| Line 1,851: | Line 1,849: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
} | } | ||
| − | # | + | #u2283_div.mouseOver { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,879: | Line 1,877: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
} | } | ||
| − | # | + | #u2283.mouseOver { |
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| − | # | + | #u2284 { |
position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 1,888: | Line 1,886: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
} | } | ||
| − | # | + | #u2285_div { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,918: | Line 1,916: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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| − | # | + | #u2285 { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,930: | Line 1,928: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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| − | # | + | #u2285_div.mouseOver { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,960: | Line 1,958: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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| − | # | + | #u2285.mouseOver { |
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| − | # | + | #u2286 { |
position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 1,969: | Line 1,967: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
} | } | ||
| − | # | + | #u2287 { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,976: | Line 1,974: | ||
height:0px; | height:0px; | ||
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| − | # | + | #u2288_div { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 1,995: | Line 1,993: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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| − | # | + | #u2288 { |
position:absolute; | position:absolute; | ||
left:1117px; | left:1117px; | ||
| Line 2,007: | Line 2,005: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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| − | # | + | #u2288_div.selected { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 2,026: | Line 2,024: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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| − | # | + | #u2288.selected { |
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position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 2,035: | Line 2,033: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
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| − | # | + | #u2290 { |
position:absolute; | position:absolute; | ||
left:717px; | left:717px; | ||
| Line 2,044: | Line 2,042: | ||
visibility:hidden; | visibility:hidden; | ||
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| − | # | + | #u2290_state0 { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 2,056: | Line 2,054: | ||
background-image:none; | background-image:none; | ||
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| − | # | + | #u2290_state0_content { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 2,063: | Line 2,061: | ||
height:1px; | height:1px; | ||
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| − | # | + | #u2291_div { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 2,092: | Line 2,090: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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| − | # | + | #u2291 { |
position:absolute; | position:absolute; | ||
left:450px; | left:450px; | ||
| Line 2,104: | Line 2,102: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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| − | # | + | #u2291_div.mouseOver { |
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left:0px; | left:0px; | ||
| Line 2,133: | Line 2,131: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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| − | # | + | #u2291.mouseOver { |
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position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 2,142: | Line 2,140: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
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position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 2,172: | Line 2,170: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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position:absolute; | position:absolute; | ||
left:300px; | left:300px; | ||
| Line 2,184: | Line 2,182: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 2,214: | Line 2,212: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 2,223: | Line 2,221: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
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position:absolute; | position:absolute; | ||
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| Line 2,251: | Line 2,249: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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position:absolute; | position:absolute; | ||
left:150px; | left:150px; | ||
| Line 2,263: | Line 2,261: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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| − | # | + | #u2295_div.mouseOver { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 2,291: | Line 2,289: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 2,300: | Line 2,298: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
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| Line 2,330: | Line 2,328: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 2,342: | Line 2,340: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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| Line 2,372: | Line 2,370: | ||
color:#FFFFFF; | color:#FFFFFF; | ||
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position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 2,381: | Line 2,379: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
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| Line 2,388: | Line 2,386: | ||
height:80px; | height:80px; | ||
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| Line 2,395: | Line 2,393: | ||
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| Line 2,404: | Line 2,402: | ||
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position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 2,418: | Line 2,416: | ||
box-shadow:none; | box-shadow:none; | ||
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| − | # | + | #u2301 { |
position:absolute; | position:absolute; | ||
left:1337px; | left:1337px; | ||
| Line 2,425: | Line 2,423: | ||
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| Line 2,433: | Line 2,431: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
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position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 2,440: | Line 2,438: | ||
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position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 2,447: | Line 2,445: | ||
height:205px; | height:205px; | ||
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position:absolute; | position:absolute; | ||
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width:202px; | width:202px; | ||
height:205px; | height:205px; | ||
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position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 2,462: | Line 2,460: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
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position:absolute; | position:absolute; | ||
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height:80px; | height:80px; | ||
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position:absolute; | position:absolute; | ||
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width:80px; | width:80px; | ||
height:80px; | height:80px; | ||
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position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 2,484: | Line 2,482: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
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| Line 2,491: | Line 2,489: | ||
height:80px; | height:80px; | ||
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position:absolute; | position:absolute; | ||
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width:80px; | width:80px; | ||
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| Line 2,506: | Line 2,504: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
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height:80px; | height:80px; | ||
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position:absolute; | position:absolute; | ||
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width:125px; | width:125px; | ||
height:80px; | height:80px; | ||
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position:absolute; | position:absolute; | ||
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| Line 2,528: | Line 2,526: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
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| Line 2,546: | Line 2,544: | ||
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position:absolute; | position:absolute; | ||
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width:171px; | width:171px; | ||
height:51px; | height:51px; | ||
| Line 2,557: | Line 2,555: | ||
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position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 2,564: | Line 2,562: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
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position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
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position:absolute; | position:absolute; | ||
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width:6px; | width:6px; | ||
height:1px; | height:1px; | ||
color:transparent; | color:transparent; | ||
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position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 2,587: | Line 2,585: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
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| Line 2,601: | Line 2,599: | ||
box-shadow:none; | box-shadow:none; | ||
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position:absolute; | position:absolute; | ||
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width:390px; | width:390px; | ||
height:116px; | height:116px; | ||
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position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 2,615: | Line 2,613: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
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text-align:right; | text-align:right; | ||
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| − | # | + | #u2319 { |
position:absolute; | position:absolute; | ||
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width:313px; | width:313px; | ||
height:51px; | height:51px; | ||
| Line 2,646: | Line 2,644: | ||
text-align:right; | text-align:right; | ||
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position:absolute; | position:absolute; | ||
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word-wrap:break-word; | word-wrap:break-word; | ||
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left:0px; | left:0px; | ||
| Line 2,660: | Line 2,658: | ||
height:80px; | height:80px; | ||
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position:absolute; | position:absolute; | ||
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width:80px; | width:80px; | ||
height:80px; | height:80px; | ||
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position:absolute; | position:absolute; | ||
left:2px; | left:2px; | ||
| Line 2,675: | Line 2,673: | ||
word-wrap:break-word; | word-wrap:break-word; | ||
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box-shadow:none; | box-shadow:none; | ||
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position:absolute; | position:absolute; | ||
left:117px; | left:117px; | ||
| Line 2,696: | Line 2,694: | ||
height:44px; | height:44px; | ||
} | } | ||
| − | # | + | #u2324 { |
position:absolute; | position:absolute; | ||
left:0px; | left:0px; | ||
| Line 2,702: | Line 2,700: | ||
width:161px; | width:161px; | ||
white-space:nowrap; | white-space:nowrap; | ||
| + | } | ||
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| + | position:absolute; | ||
| + | left:0px; | ||
| + | top:0px; | ||
| + | width:1200px; | ||
| + | height:38px; | ||
| + | background:inherit; | ||
| + | background-color:rgba(255, 255, 255, 0); | ||
| + | border:none; | ||
| + | border-radius:0px; | ||
| + | -moz-box-shadow:none; | ||
| + | -webkit-box-shadow:none; | ||
| + | box-shadow:none; | ||
| + | } | ||
| + | #u2325 { | ||
| + | position:absolute; | ||
| + | left:117px; | ||
| + | top:943px; | ||
| + | width:1200px; | ||
| + | height:38px; | ||
| + | } | ||
| + | #u2326 { | ||
| + | position:absolute; | ||
| + | left:0px; | ||
| + | top:0px; | ||
| + | width:1200px; | ||
| + | word-wrap:break-word; | ||
| + | } | ||
| + | #u2327_div { | ||
| + | position:absolute; | ||
| + | left:0px; | ||
| + | top:0px; | ||
| + | width:1200px; | ||
| + | height:32px; | ||
| + | background:inherit; | ||
| + | background-color:rgba(255, 255, 255, 0); | ||
| + | border:none; | ||
| + | border-radius:0px; | ||
| + | -moz-box-shadow:none; | ||
| + | -webkit-box-shadow:none; | ||
| + | box-shadow:none; | ||
| + | } | ||
| + | #u2327 { | ||
| + | position:absolute; | ||
| + | left:117px; | ||
| + | top:990px; | ||
| + | width:1200px; | ||
| + | height:32px; | ||
| + | } | ||
| + | #u2328 { | ||
| + | position:absolute; | ||
| + | left:0px; | ||
| + | top:0px; | ||
| + | width:1200px; | ||
| + | word-wrap:break-word; | ||
} | } | ||
| + | |||
| + | |||
</style> | </style> | ||
| Line 2,742: | Line 2,798: | ||
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<script type="text/javascript"> | <script type="text/javascript"> | ||
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| − | + | var _creator = function() { return 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| |
return _creator(); | return _creator(); | ||
})()); | })()); | ||
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</script> | </script> | ||
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<body> | <body> | ||
| − | + | <div id="base" class=""> | |
| − | + | ||
<!-- Unnamed (矩形) --> | <!-- Unnamed (矩形) --> | ||
| − | <div id=" | + | <div id="u2223" class="ax_default _标题_2"> |
| − | <div id=" | + | <div id="u2223_div" class=""></div> |
<!-- Unnamed () --> | <!-- Unnamed () --> | ||
| − | <div id=" | + | <div id="u2224" class="text" style="visibility: visible;"> |
<p><span>Cell culture</span></p> | <p><span>Cell culture</span></p> | ||
</div> | </div> | ||
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<!-- Unnamed (矩形) --> | <!-- Unnamed (矩形) --> | ||
| − | <div id=" | + | <div id="u2225" class="ax_default _正文"> |
| − | <div id=" | + | <div id="u2225_div" class=""></div> |
<!-- Unnamed () --> | <!-- Unnamed () --> | ||
| − | <div id=" | + | <div id="u2226" class="text" style="visibility: visible;"> |
<p><span>Bl21 cells were cultured 12 h at 37 °C with shaking in 50ml Centrifuge tube with 10ml LB medium containing the appropriate antibiotic. These cultures were used to inoculate 100 mL of LB medium containing the appropriate antibiotics in a 500 mL Erlenmeyer flask at an initial OD600 of 0.6-0.8. To induce the expression of the dsz genes under the Ptac or Plac or PT7 promoter, 1 mM IPTG was added to the medium. Cultures were incubated 12h at 16 °C. </span></p> | <p><span>Bl21 cells were cultured 12 h at 37 °C with shaking in 50ml Centrifuge tube with 10ml LB medium containing the appropriate antibiotic. These cultures were used to inoculate 100 mL of LB medium containing the appropriate antibiotics in a 500 mL Erlenmeyer flask at an initial OD600 of 0.6-0.8. To induce the expression of the dsz genes under the Ptac or Plac or PT7 promoter, 1 mM IPTG was added to the medium. Cultures were incubated 12h at 16 °C. </span></p> | ||
</div> | </div> | ||
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<!-- Unnamed (矩形) --> | <!-- Unnamed (矩形) --> | ||
| − | <div id=" | + | <div id="u2227" class="ax_default _标题_2"> |
| − | <div id=" | + | <div id="u2227_div" class=""></div> |
<!-- Unnamed () --> | <!-- Unnamed () --> | ||
| − | <div id=" | + | <div id="u2228" class="text" style="visibility: visible;"> |
<p><span>Whole cell catalysis</span></p> | <p><span>Whole cell catalysis</span></p> | ||
</div> | </div> | ||
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<!-- Unnamed (矩形) --> | <!-- Unnamed (矩形) --> | ||
| − | <div id=" | + | <div id="u2229" class="ax_default _正文"> |
| − | <div id=" | + | <div id="u2229_div" class=""></div> |
<!-- Unnamed () --> | <!-- Unnamed () --> | ||
| − | <div id=" | + | <div id="u2230" class="text" style="visibility: visible;"> |
<p><span>Cells were harvested by centrifugation, washed twice with 0.9% PBS, and the cell pellet was resuspended in 20 mM PBS buffer at an OD600 of 2.0 in a 100 mL Erlenmeyer flask. This resting cells preparation was used for the BDS assays, which were carried out in an orbital shaker at 30 °C and 200 rpm after adding 4S compounds (DBT). Samples were collected periodically and, unless otherwise specified, extracted with an equal volume of acetonitrile for HPLC analysis of substrate consumed and product(s) generated after centrifugation at 14000*g for 10 min. To check the extracellular location of some 4S-pathway intermediates, resting cells were centrifugedat 14000*g for 10 min and the supernatant was extracted with acetonitrile and analyzed by HPLC. Three/four different biological samples were used to calculate substrate consumption and intermediates/product formation in all experiments reported in this work. </span></p> | <p><span>Cells were harvested by centrifugation, washed twice with 0.9% PBS, and the cell pellet was resuspended in 20 mM PBS buffer at an OD600 of 2.0 in a 100 mL Erlenmeyer flask. This resting cells preparation was used for the BDS assays, which were carried out in an orbital shaker at 30 °C and 200 rpm after adding 4S compounds (DBT). Samples were collected periodically and, unless otherwise specified, extracted with an equal volume of acetonitrile for HPLC analysis of substrate consumed and product(s) generated after centrifugation at 14000*g for 10 min. To check the extracellular location of some 4S-pathway intermediates, resting cells were centrifugedat 14000*g for 10 min and the supernatant was extracted with acetonitrile and analyzed by HPLC. Three/four different biological samples were used to calculate substrate consumption and intermediates/product formation in all experiments reported in this work. </span></p> | ||
</div> | </div> | ||
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<!-- Unnamed (矩形) --> | <!-- Unnamed (矩形) --> | ||
| − | <div id=" | + | <div id="u2231" class="ax_default _标题_2"> |
| − | <div id=" | + | <div id="u2231_div" class=""></div> |
<!-- Unnamed () --> | <!-- Unnamed () --> | ||
| − | <div id=" | + | <div id="u2232" class="text" style="visibility: visible;"> |
<p><span>Analytical methods</span></p> | <p><span>Analytical methods</span></p> | ||
</div> | </div> | ||
| Line 2,884: | Line 2,888: | ||
<!-- Unnamed (矩形) --> | <!-- Unnamed (矩形) --> | ||
| − | <div id=" | + | <div id="u2233" class="ax_default _正文"> |
| − | <div id=" | + | <div id="u2233_div" class=""></div> |
<!-- Unnamed () --> | <!-- Unnamed () --> | ||
| − | <div id=" | + | <div id="u2234" class="text" style="visibility: visible;"> |
<p><span>HPLC was employed to analyze the concentration of the 4S compounds (DBT, DBTO2,and HBP) employing a C18 column; the mobile phase was a mixture acetonitrile/water (70:30) at 0.7mL min"1 flow rate. Peaks were monitored at 280nm wave lengths. Calibrations were performed using highly purified standards of each compound</span></p> | <p><span>HPLC was employed to analyze the concentration of the 4S compounds (DBT, DBTO2,and HBP) employing a C18 column; the mobile phase was a mixture acetonitrile/water (70:30) at 0.7mL min"1 flow rate. Peaks were monitored at 280nm wave lengths. Calibrations were performed using highly purified standards of each compound</span></p> | ||
</div> | </div> | ||
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<!-- Unnamed (组合) --> | <!-- Unnamed (组合) --> | ||
| − | <div id=" | + | <div id="u2236" class="ax_default"> |
<!-- Unnamed (矩形) --> | <!-- Unnamed (矩形) --> | ||
| − | <div id=" | + | <div id="u2237" class="ax_default box_1"> |
| − | <div id=" | + | <div id="u2237_div" class=""></div> |
<!-- Unnamed () --> | <!-- Unnamed () --> | ||
| − | <div id=" | + | <div id="u2238" class="text" style="visibility: visible;"> |
<p><span>Home</span></p> | <p><span>Home</span></p> | ||
</div> | </div> | ||
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<!-- project (组合) --> | <!-- project (组合) --> | ||
| − | <div id=" | + | <div id="u2239" class="ax_default" data-label="project"> |
<!-- Unnamed (矩形) --> | <!-- Unnamed (矩形) --> | ||
| − | <div id=" | + | <div id="u2240" class="ax_default box_1"> |
| − | <div id=" | + | <div id="u2240_div" class=""></div> |
<!-- Unnamed () --> | <!-- Unnamed () --> | ||
| − | <div id=" | + | <div id="u2241" class="text" style="visibility: visible;"> |
<p><span>Project</span></p> | <p><span>Project</span></p> | ||
</div> | </div> | ||
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<!-- Project (动态面板) --> | <!-- Project (动态面板) --> | ||
| − | <div id=" | + | <div id="u2242" class="ax_default ax_default_hidden" data-label="Project" style="display: none; visibility: hidden"> |
| − | <div id=" | + | <div id="u2242_state0" class="panel_state" data-label="State1"> |
| − | <div id=" | + | <div id="u2242_state0_content" class="panel_state_content"> |
<!-- Design&Result (矩形) --> | <!-- Design&Result (矩形) --> | ||
| − | <div id=" | + | <div id="u2243" class="ax_default box_1 ax_default_hidden" data-label="Design&Result" style="display: none; visibility: hidden"> |
| − | <div id=" | + | <div id="u2243_div" class=""></div> |
<!-- Unnamed () --> | <!-- Unnamed () --> | ||
| − | <div id=" | + | <div id="u2244" class="text" style="visibility: visible;"> |
| − | <p><span>Design& | + | <p><span>Design&Results</span></p> |
</div> | </div> | ||
</div> | </div> | ||
<!-- 4S-Pathway (矩形) --> | <!-- 4S-Pathway (矩形) --> | ||
| − | <div id=" | + | <div id="u2245" class="ax_default box_1 ax_default_hidden" data-label="4S-Pathway" style="display: none; visibility: hidden"> |
| − | <div id=" | + | <div id="u2245_div" class=""></div> |
<!-- Unnamed () --> | <!-- Unnamed () --> | ||
| − | <div id=" | + | <div id="u2246" class="text" style="visibility: visible;"> |
<p><span>4S-Pathway</span></p> | <p><span>4S-Pathway</span></p> | ||
</div> | </div> | ||
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<!-- Background (矩形) --> | <!-- Background (矩形) --> | ||
| − | <div id=" | + | <div id="u2247" class="ax_default box_1 ax_default_hidden" data-label="Background" style="display: none; visibility: hidden"> |
| − | <div id=" | + | <div id="u2247_div" class=""></div> |
<!-- Unnamed () --> | <!-- Unnamed () --> | ||
| − | <div id=" | + | <div id="u2248" class="text" style="visibility: visible;"> |
<p><span>Background</span></p> | <p><span>Background</span></p> | ||
</div> | </div> | ||
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<!-- Overview (矩形) --> | <!-- Overview (矩形) --> | ||
| − | <div id=" | + | <div id="u2249" class="ax_default box_1 ax_default_hidden" data-label="Overview" style="display: none; visibility: hidden"> |
| − | <div id=" | + | <div id="u2249_div" class=""></div> |
<!-- Unnamed () --> | <!-- Unnamed () --> | ||
| − | <div id=" | + | <div id="u2250" class="text" style="visibility: visible;"> |
<p><span>Overview</span></p> | <p><span>Overview</span></p> | ||
</div> | </div> | ||
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<!-- modeling (组合) --> | <!-- modeling (组合) --> | ||
| − | <div id=" | + | <div id="u2251" class="ax_default" data-label="modeling"> |
<!-- Unnamed (矩形) --> | <!-- Unnamed (矩形) --> | ||
| − | <div id=" | + | <div id="u2252" class="ax_default box_1"> |
| − | <div id=" | + | <div id="u2252_div" class=""></div> |
<!-- Unnamed () --> | <!-- Unnamed () --> | ||
| − | <div id=" | + | <div id="u2253" class="text" style="visibility: visible;"> |
<p><span>Modeling</span></p> | <p><span>Modeling</span></p> | ||
</div> | </div> | ||
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<!-- Modeling (动态面板) --> | <!-- Modeling (动态面板) --> | ||
| − | <div id=" | + | <div id="u2254" class="ax_default ax_default_hidden" data-label="Modeling" style="display: none; visibility: hidden"> |
| − | <div id=" | + | <div id="u2254_state0" class="panel_state" data-label="State1"> |
| − | <div id=" | + | <div id="u2254_state0_content" class="panel_state_content"> |
<!-- Water-oil ratio (矩形) --> | <!-- Water-oil ratio (矩形) --> | ||
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<p><span>Water-oil ratio</span></p> | <p><span>Water-oil ratio</span></p> | ||
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<p><span>Enzymes ratio</span></p> | <p><span>Enzymes ratio</span></p> | ||
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| − | <p style="font-size:36px;"><span style="font-family:'Comic Sans MS Normal', 'Comic Sans MS';font-weight:400;"> | + | <p style="font-size:36px;"><span style="font-family:'Comic Sans MS Normal', 'Comic Sans MS';font-weight:400;">Sulfur Killer</span><span style="font-family:'Comic Sans MS Negreta', 'Comic Sans MS Normal', 'Comic Sans MS';font-weight:700;font-size:28px;"></span></p><p style="font-size:28px;"><span style="font-family:'Comic Sans MS Negreta', 'Comic Sans MS Normal', 'Comic Sans MS';font-weight:700;">wiki group suport</span><span style="font-family:'Arial Normal', 'Arial';font-weight:400;"> </span></p> |
</div> | </div> | ||
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<p><span>South China University of tecnology</span></p><p><span>GuangZhou,China</span></p> | <p><span>South China University of tecnology</span></p><p><span>GuangZhou,China</span></p> | ||
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<p><span>Protocols</span></p> | <p><span>Protocols</span></p> | ||
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| + | <p><span>DszB in cell extracts</span></p> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | <!-- Unnamed (矩形) --> | ||
| + | <div id="u2327" class="ax_default _正文"> | ||
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| + | <div id="u2328" class="text" style="visibility: visible;"> | ||
| + | <p><span>For the addition using cell extracts, the recombinant strain BL21-dszB cells were grown as detailed above (Cell culture), washed twice with 0.9% NaCl and resuspended in 50 mM HEPES buffer pH 8.0 at an OD600 of 20, and disrupted by passage through an ultrasonic fragmentation 2h. Cell debris were removed by centrifugation at 16,000 g for 20 min at 4 °C, and the resulting supernatant was used as cell extract. Addition of crude cell extracts containing DszB (0.5 mg protein/mL) will be added to our desulfurization system.</span></p> | ||
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Latest revision as of 22:16, 19 October 2016
Bl21 cells were cultured 12 h at 37 °C with shaking in 50ml Centrifuge tube with 10ml LB medium containing the appropriate antibiotic. These cultures were used to inoculate 100 mL of LB medium containing the appropriate antibiotics in a 500 mL Erlenmeyer flask at an initial OD600 of 0.6-0.8. To induce the expression of the dsz genes under the Ptac or Plac or PT7 promoter, 1 mM IPTG was added to the medium. Cultures were incubated 12h at 16 °C.
Whole cell catalysis
Cells were harvested by centrifugation, washed twice with 0.9% PBS, and the cell pellet was resuspended in 20 mM PBS buffer at an OD600 of 2.0 in a 100 mL Erlenmeyer flask. This resting cells preparation was used for the BDS assays, which were carried out in an orbital shaker at 30 °C and 200 rpm after adding 4S compounds (DBT). Samples were collected periodically and, unless otherwise specified, extracted with an equal volume of acetonitrile for HPLC analysis of substrate consumed and product(s) generated after centrifugation at 14000*g for 10 min. To check the extracellular location of some 4S-pathway intermediates, resting cells were centrifugedat 14000*g for 10 min and the supernatant was extracted with acetonitrile and analyzed by HPLC. Three/four different biological samples were used to calculate substrate consumption and intermediates/product formation in all experiments reported in this work.
Analytical methods
HPLC was employed to analyze the concentration of the 4S compounds (DBT, DBTO2,and HBP) employing a C18 column; the mobile phase was a mixture acetonitrile/water (70:30) at 0.7mL min"1 flow rate. Peaks were monitored at 280nm wave lengths. Calibrations were performed using highly purified standards of each compound
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Protocols
DszB in cell extracts
For the addition using cell extracts, the recombinant strain BL21-dszB cells were grown as detailed above (Cell culture), washed twice with 0.9% NaCl and resuspended in 50 mM HEPES buffer pH 8.0 at an OD600 of 20, and disrupted by passage through an ultrasonic fragmentation 2h. Cell debris were removed by centrifugation at 16,000 g for 20 min at 4 °C, and the resulting supernatant was used as cell extract. Addition of crude cell extracts containing DszB (0.5 mg protein/mL) will be added to our desulfurization system.
