ࡱ> 02/O H bjbjee .ee\     !!!8Ym!9 ($)"$p   >>>  >>>:h,̆TR  09 RO%pO%O% >L9 O% : SYTOX staining for yeast flow This is a replacement for the old propidium iodide staining CARE! This protocol uses large amounts of RNAse A. To protect your future RNA experiments, take the following precautions: Use our designated RNase pipettes to handle RNAse A solutions Use filter tips to handle RNase A stock solution Spin tubes containing RNase A solutions before opening Discard RNAse A waste and contaminated plasticware directly into waste bin - not your tip waste or a beaker that may be reused Treat RNase A stock like it is toxic even if it isnt Resuspend 1-10 million yeast cells in 300 l PBST, add 4 l 20 mg/ml RNase A Incubate overnight at 37C Pulse to 10k to pellet cells, remove RNase A solution Wash cells with 300 l PBST Resuspend cells in 100-1000 l of PBST (depending on how many cells stained) + 1 M SYTOX (from 5 mM stock in DMSO, so 1:5,000) Let cells stain overnight at 4C or for 1 hour at room temperature Run directly on flow * NOTE: If the samples are going to run on the ImageStream, this protocol will generate overstained samples. Either reduce the staining (i.e. use less Sytox, shorter incubation), or run the samples on a conventional flow cytometer such as the Fortessa. PBST: PBS + 0.01% Triton X100      FILENAME SYTOX staining for yeast flow.doc v1.0 Houseley lab  PAGE 1 ^ _ m n    ? C D k l s  #   $ % 3 7 : ӼӸᰩh%hCmHnHuh(jh(Uh}jh}U h)h)h)h:hy6hy6hy65B*phhPOJQJ^JhPhCOJQJ^JhChMh)5hM5CJ(aJ(hC5CJ(aJ(2[\ F } 2 3 4 r s ^gdCgdC & FgdCgd)$a$gd) 8 9 E F G H ^gdy6: ; A B C D F G H h)h)h}h(h0JmHnHu h(0Jjh(0JU,1h. A!"#$% w2 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@6666_HmH nH sH tH @`@ NormalCJ_HaJmH sH tH DA`D Default Paragraph FontRiR  Table Normal4 l4a (k (No List 4@4 Mr2Header  9r 4 @4 Mr2Footer  9r .)@. Mr2 Page NumberPK![Content_Types].xmlN0EH-J@%ǎǢ|ș$زULTB l,3;rØJB+$G]7O٭VGRU1a$N% ʣꂣKЛjVkUDRKQj/dR*SxMPsʧJ5$4vq^WCʽ D{>̳`3REB=꽻Ut Qy@֐\.X7<:+& 0h @>nƭBVqu ѡ{5kP?O&Cנ Aw0kPo۵(h[5($=CVs]mY2zw`nKDC]j%KXK 'P@$I=Y%C%gx'$!V(ekڤք'Qt!x7xbJ7 o߼W_y|nʒ;Fido/_1z/L?>o_;9:33`=—S,FĔ觑@)R8elmEv|!ո/,Ә%qh|'1:`ij.̳u'k CZ^WcK0'E8S߱sˮdΙ`K}A"NșM1I/AeހQתGF@A~eh-QR9C 5 ~d"9 0exp<^!͸~J7䒜t L䈝c\)Ic8E&]Sf~@Aw?'r3Ȱ&2@7k}̬naWJ}N1XGVh`L%Z`=`VKb*X=z%"sI<&n| .qc:?7/N<Z*`]u-]e|aѸ¾|mH{m3CԚ .ÕnAr)[;-ݑ$$`:Ʊ>NVl%kv:Ns _OuCX=mO4m's߸d|0n;pt2e}:zOrgI( 'B='8\L`"Ǚ 4F+8JI$rՑVLvVxNN";fVYx-,JfV<+k>hP!aLfh:HHX WQXt,:JU{,Z BpB)sֻڙӇiE4(=U\.O. +x"aMB[F7x"ytѫиK-zz>F>75eo5C9Z%c7ܼ%6M2ˊ 9B" N "1(IzZ~>Yr]H+9pd\4n(Kg\V$=]B,lוDA=eX)Ly5ot e㈮bW3gp : j$/g*QjZTa!e9#i5*j5ö fE`514g{7vnO(^ ,j~V9;kvv"adV݊oTAn7jah+y^@ARhW.GMuO "/e5[s󿬅`Z'WfPt~f}kA'0z|>ܙ|Uw{@՘tAm'`4T֠2j ۣhvWwA9 ZNU+Awvhv36V`^PK! ѐ'theme/theme/_rels/themeManager.xml.relsM 0wooӺ&݈Э5 6?$Q ,.aic21h:qm@RN;d`o7gK(M&$R(.1r'JЊT8V"AȻHu}|$b{P8g/]QAsم(#L[PK-![Content_Types].xmlPK-!֧6 0_rels/.relsPK-!kytheme/theme/themeManager.xmlPK-!g theme/theme/theme1.xmlPK-! ѐ' theme/theme/_rels/themeManager.xml.relsPK] H NN[[[^: H   H  9OVX^!8@0(  B S  ?#chFI9FI#C79:DI#CI ~KE4.AP^`OJPJQJ^Jo(-^`OJQJ^Jo(hHop^p`OJQJo(hH@ ^@ `OJQJo(hH^`OJQJ^Jo(hHo^`OJQJo(hH^`OJQJo(hH^`OJQJ^Jo(hHoP^P`OJQJo(hHh^`OJQJo(hHh^`OJQJ^Jo(hHohp^p`OJQJo(hHh@ ^@ `OJQJo(hHh^`OJQJ^Jo(hHoh^`OJQJo(hHh^`OJQJo(hHh^`OJQJ^Jo(hHohP^P`OJQJo(hHE4.A N                  '&Mmp.C%`4'*Mr2; BqBcH7TYs,[QycRdisfgxomu./|}fL0(sFu7bRin(:)y6Pd@###H@UnknownG.[x Times New Roman5Symbol3. .[x ArialC.,.{$ Calibri Light7..{$ Calibri?= .Cx Courier New;WingdingsA$BCambria Math"qhNëg00qr4 3qHP ?72!xxL  Prepare 0jhouseleHanane Hadj-Moussa  Oh+'0   @ L Xdlt| Prepare 0 jhousele Normal.dotmHanane Hadj-Moussa6Microsoft Office Word@`4<@@pT0 ՜.+,0 hp  University of Edinburgh  Prepare 0 Title  !"#$%&()*+,-.1Root Entry FцT31Table c%WordDocument.SummaryInformation(DocumentSummaryInformation8'CompObjr  F Microsoft Word 97-2003 Document MSWordDocWord.Document.89q