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Last modified
7/14/2009 5:02:30 PM
Creation date
5/20/2009 3:04:31 PM
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UCREFRP
UCREFRP Catalog Number
7717
Author
Rosenfeld, M. J.
Title
Genetic Analysis of the Colorado River Gila
USFW Year
n.d.
USFW - Doc Type
Non-fatal Sampling, Cell Culture, Chromosomal Analysis and Electrophoresis.
Copyright Material
NO
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<br />tion at 43 aC. <br /> <br />Silve~-stained ch~omosomes we~e counte~stained <br /> <br />with 21. Giemsa made up in 0.15 M So~ensen buffe~ at pH 6.8 fio~ <br /> <br />75-90 sec. Finished slides f~om both p~ocedu~es we~e ~insed wiith <br /> <br />distilled wate~, <br /> <br />blotted with bibulous pape~, <br /> <br />ai~ d~ied and <br /> <br />placed in xylene fo~ 15 min p~io~ to mounting with Pe~mount ~nd <br /> <br />no. 1 cove~slips. <br /> <br />Cells we~e obse~ved with a Zeiss Photomic~oscope III usinq a <br /> <br />100 X planoch~omatic objective. <br /> <br />Ch~omosome sp~eads we~e pho~o- <br /> <br />g~aphed th~ough filte~s, <br /> <br />g~een fo~ g~oss-stained chromos~me <br /> <br />sp~eads and yellow fo~ silve~-stained ones. <br /> <br />Kodak Technical ~an <br /> <br />film was exposed at ASA 20 fo~ g~oss stained p~epa~ations ~nd <br /> <br />developed fo~ 9 min in a 501. solution of Kodak 076 developer at <br /> <br />23 ac; at ASA 12.5 fo~ silve~-stained ones and developed for 5.5 <br /> <br />.min in Ethol Tech develope~ at 21 aC. <br /> <br />Prints we~e made on K~dak <br />! <br /> <br />RC paper using Dekt~l" developer, from which sorted karyot~pes <br /> <br />. <br />were prepared. Chromosomes were" measured on a digitizer usin~ <br /> <br />>:.~,' <br /> <br />Apple II computer. <br /> <br />~<,' {-, <br /> <br />Electrophoresis: <br /> <br />Approxjmately 1 ml of bl60d was taken .rom <br /> <br />each fish through caudal vein puncture using a 27g needle andia 1 <br /> <br />ml <br /> <br />tuberculin syringe. <br /> <br />Clotting was <br /> <br />inhibited with cittate <br />I <br /> <br />anticoagulant and samples were kept on ice for as long as 2 d <br /> <br />p~ior to processing. <br /> <br />Afte~ cent~ifuging whole blood at 300 g <br /> <br />and 4 DC for <br /> <br />supernatant seram was removed and frozen <br /> <br />15 min, <br /> <br />at -80 ClC. <br /> <br />The red blood cells we~e resuspended and washed three <br /> <br />times in 0.81. sodium chlo~ide. <br /> <br />Cent~ifugations between ~ach <br /> <br />ridrl j t j nfl rd <br /> <br />S d] i rl L: W p r. E' d CJ Ii C un del' <br /> <br />the <"=>c~,fTiC' cCJfldit.lDrl<;:; a~;: fOf <br /> <br />; , <br /> <br />i <br /> <br />~ <br /> <br />
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