ࡱ> 685c bjbj** .&HSbHSbJ R::8,.$RR(zzzUUUgiiiiii$ vUUUUUzzgggUzzggUggggz`hf|gS0g !} !g !gUUgUUUUUTUUUUUUU !UUUUUUUUU:B |: Yeast RNA miniprep This protocol is optimised for high throughput, but there are a few optional steps to get cleaner RNA. Without optional steps, RNA is good for agarose northerns and RT-PCR, for acrylamide northerns the longer prep can help. Harvest 2x107 cells according to separate cell harvesting protocol, 2ml tubes containing cell pellet should be kept on ice, and thawing the pellet is unnecessary. In our hands yields are more reproducible starting from pellets frozen on N2 than from ethanol-fixed cells. If using ethanol fixed cells, wash the pellet once with cold water. Make GTC phenol: 1:1 ratio GTC mix : phenol pH7, you need 640 l per sample Add 50(l-ish of glass beads and 40(l GTC phenol, vortex for 5 minutes at 4( On ice, add 600(l GTC phenol, vortex briefly Incubate at 65( for 10 min, then place on ice for 10 min Add 300(l chloroform/IAA then 160(l 100mM NaOAc pH5.2, vortex briefly Spin 5 min at 14,000rpm in a microfuge in the fume hood For cleaner RNA, skip to the next page, or proceed below for quick prep Remove 500(l upper phase and add to 1ml ethanol in a new tube, vortex briefly Spin 15min at 14,000rpm 4(C, pour off supernatant into phenol waste Add 1ml 70% ethanol, vortex well Spin 1 minute at 14,000rpm, pour off supernatant Spin 30s at 14,000rpm, pipette off remaining supernatant Dry briefly (30s-2min) then pipette 7(l water onto pellet Vortex briefly, incubate at room temperature for 10 minutes Spin sample 30s at 14,000rpm before use Run 3(l per lane on an agarose northern Cleaner RNA protocol: Add 500l upper phase to 500(l Phenol:Chloroform:IAA Spin 1 minute at 14,000rpm in a microfuge in the fume hood Remove 450(l upper phase, add to 500(l Chloroform:IAA Spin 1 minute at 14,000rpm in a microfuge in the fume hood Remove 400(l upper phase, and add to 1ml ethanol Spin 15min at 14,000rpm 4(C, pour off supernatant Add 1ml 70% ethanol, vortex well Spin 1 minute at 14,000rpm, pour off supernatant, spin again, pipette off the rest Dry briefly (30s-2min) then re-suspend pellet in 6.5(l water per 1 OD cells Dilute 1:10 and quantify on Nanodrop in RNA mode Run 10(g per lane on a northern Solutions GTC mix: 100g GTC 100ml H2O 10.6ml 1M Na citrate pH7 4.24ml 0.5M EDTA pH8 10 minutes at 60-70(, swirl to dissolve Add 2.1 ml (-mercaptoethanol 21.2ml 20% sarkosyl This keeps for years at 4(, even if it turns a bit yellow Before use, make a 1:1 mix with phenol pH 7 phenol / chloroform / IAA: 25 parts phenol pH 7 24 parts Chloroform 1 part isoamyl alcohol chloroform / IAA: 24 parts Chloroform : 1 part isoamyl alcohol      FILENAME Yeast RNA miniprep.doc v4.1 Houseley lab  PAGE 1 {   9 = d h i o  G H     ƽ׽祡祽 jmh8r=h=2hsb jh8r=h8r=ho^h+hMp jmh81 hvPh!h!OJQJ^Jh! hvPH*hvPheh} hz H*hNBhh`h ih81hO9h8r=5h8r=h8r=5CJ(aJ(2H I Z   J K    d gdo^`gdHcgdHcgd=2`gd!$a$gd8r=  > R S d i l m o u z     ! . E S c d e ~   6 N S T ] ^ ֽ֦ jmhh!hhG jho^ho^h+he jmh_ h_ hNBhHchHc6hHcho^6 hNB6hHchhO9h ihLE jmh8r=hsb jh8r=h8r=4d e 7 8 r s PQgd_ `gdsbgdsbgdo^ "#%478  01KL (XYZ[`akyz{|߿߿߿ h8r=H*h!hHch+5hHchHc5h8r= jmh ih ih=2huhO9 jhHch+ jmh_ jmhsbh;OJQJ^Jh;hsbh_ hHchHc6 hNB6 hO96hHc jmhHc212de(YZz{| *Bgd8r=gd=2gdHc`gd_   -.AB]^   IJKMNPQSTVWabxy~jh`0JUhXUKhzCch`mHnHuh`jh`Uh@ jh@ U h8r=h=2 h8r=hLEhehe*hLE jh!h! jbh8r=hHch jh8r=hnh8r=h=23B  JLMOPRSUV@ ^@ gde p^p`gdegdLEgd8r= h8r=h=2h@ h`jh`0JU*hzCc0JmHnHu*jh`0JU h`0J,1h. A!"#$% s2 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@_HmH nH sH tH @`@ NormalCJ_HaJmH sH tH DA`D Default Paragraph FontRiR  Table Normal4 l4a (k (No List 4@4 o^Header  9r 4 @4 o^Footer  9r .)@. o^ Page Number>"> o^ Footnote TextCJaJ@& 1@ o^Footnote ReferenceH*PK![Content_Types].xmlN0EH-J@%ǎǢ|ș$زULTB l,3;rØJB+$G]7O٭Vc:E3v@P~Ds |w< #& DDQQQT  d B  .ELNT!8@0(  B S  ? :ODL9 A  : A J L M O P R S U V hm@O  J L M O P R S U V 33333..Ec!6T% (XA I J J M M V ..Ec!6T% (XA I 54[jz _ A?xe|:W$(N(e*i`/8r=LEG~GXUKvPUs,[o^5`HczCc in#x~Mp5%7}fyZ81sb;`3NBR+!O9 }xu`=2@ J L @ @UnknownG.[x Times New Roman5Symbol3. .[x ArialA$BCambria Math"1hצ)ۈ} &} &!4@ @ 2qHP ?8r=2!xxGg Yeast RNA miniprepjhousele Jon Houseley Oh+'0 $ D P \hpxYeast RNA miniprep jhousele Normal.dotmJon Houseley15Microsoft Office Word@J6@ze @uf|} ՜.+,0 hp  University of Edinburgh& @  Yeast RNA miniprep Title  !"#$&'()*+,./012347Root Entry FLif|91Table !WordDocument.&SummaryInformation(%DocumentSummaryInformation8-CompObjr  F Microsoft Word 97-2003 Document MSWordDocWord.Document.89q