ࡱ> /1. bjbjww .^k^kC8%p99(aaa<<<TVVVVVV!dV<<<<<Vaak<RaaT<Ta0-@0;v;;<<<<<<<VVp<<<<<<<;<<<<<<<<< : Phenol:chloroform extraction and precipitation This is a very standard purification method for DNA and RNA Firstly know your phenol:chloroform. pH matters. At pH 7, genomic DNA is removed in the extraction, at pH 8 genomic DNA is retained. RNA and small DNA fragments are always retained. Phenol:chloroform is sold as a 25:24:1 mixture with isoamyl alcohol, pre-saturated with Tris, usually at pH8. This is labelled as Phenol:chloroform pH8, BUT IT ISNT! Phenol is acidic so phenol saturated with Tris pH8 ends up as pH7. For pH8, buy the phenol that comes with a pH10 saturation buffer add this when you open the bottle, shake really well then leave over-night to settle Phenol:chloroform stocks always have a Tris buffer which forms a layer on the top. When taking phenol:chloroform from the bottle ALWAYS TAKE THE BOTTOM LAYER. Otherwise you just get Tris! Phenol:chloroform is toxic and corrosive, use in the fume hood, wear gloves and eye protection. Add 1 volume of phenol:chloroform pH8 (or pH7 for RNA) to your sample, shake really well then centrifuge top speed for 1 minute at room temperature. Look at the sample it should be in two layers (phases) usually with some crud at the interphase. If the interphase is not tight then re-centrifuge for 5-10 minutes. Ie: for 100l reaction, add 100l phenol:chloroform Pipette the aqueous phase (top layer) into a new tube, being careful not to take the interphase. If you make a mess of this, put everything back in the first tube, re-centrifuge and try again. Dispose of the organic phase (the bottom layer) to the phenol:chloroform waste bottle To the aqueous phase, add 1l of glycogen, 10% volume 3M NaOAc and 2-2.5 volumes 100% ethanol, vortex well Ie: for 100l add 1l glycogen, 10l 3M NaOAc and 250l ethanol Spin top speed for 15 minutes at 4 - a pellet should be visible Spin at room temperature if there is a lot of SDS in the sample Pour off the liquid, add 3 volumes 70% ethanol, invert to mix, spin 30s top speed Pour off the liquid, pulse to top speed Remove liquid with pipette, let pellet dry for ~5 minutes Add required volume 10mM Tris pH8 or 0.1x TE to dissolve DNA Use water for RNA      FILENAME name.doc v1.0 Houseley lab  PAGE 1 ./0lmw e f    $ % 9 : F _  ,RSVlno@A޻ޭޭޭޭޭ޴hU&h% hU&hO hU&hU& hU&hhU&hAVB*ph hU&h)hU&hAV5\ hU&hAVhMh)5hM5CJ(aJ(h[5CJ(aJ(A/0lm e f  9 : RSAgd)$a$gd)9:vw{һҦh7h0JmHnHu h(0Jjh(0JUh%h(mHnHuh(jh(Uh jh UhU&hU&OJQJhU& hU&hU&'9:w`gdU&,1h. A!"#$% x2 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@66666_HmH nH sH tH @`@ NormalCJ_HaJmH sH tH DA`D Default Paragraph FontRiR  Table Normal4 l4a (k (No List 4@4 Mr2Header  9r 4 @4 Mr2Footer  9r .)@. Mr2 Page NumberPK![Content_Types].xmlN0EH-J@%ǎǢ|ș$زULTB l,3;rØJB+$G]7O٭Vzt7.??Źڨd/L+[梃 _¯UjyA_/֖j-A %4\/%ި+K|h(Kmgk):b| H$Y 9DZ.6)A2&`RV!*_[`dI+^DL-)>qfoΟ~_'ҨrQrW_ƿw!pƦ7/_zqԵbp@m ?ItDl,(CjLQc׎O9!N@Iw{Xe3b|`:2˭A9Tn$ءy@Q&Q3,;sg8v%= CN4I ~> zHzw0u $J}*;(wL|$C޳q-!1,hEX>ZN yݾKt l&;HPm%6sq !&}] ~@Lw?%q d98@7C̜mia_Y㩓b8F vB{cNQqsu;6~@VƾzXU+f:OllD,E|=mVePاcGxe_+gj=HSԳ;{yи½|mHmA`0D_cU\+w/ 9MOJK:WnǯIUsffn`<"K*2:&w4]3۸cB/br;}̸uF̘G}ҙ3>-=k&ڂE%睸 |Te6pp1GZ&L~AdNPjĢP gFFz٫[ euRTVxC'c*ibQTz:"dC%1!4ZŲR?rՔ)Zf\5GμMO20V4'|:xt&Ԯiv +nD?TWq]_7.u)S 4?⦾@3;S,8;^K=7>̌cC ^$77&%Bn"cy@I K?L'CWc%WSc#^w}ܓ߭ei)$ Z`%v]:Blqwae&De*2JǐYG4OPQRlnຠtSi832IQ QrZ.afٽ\HYʚU6iaT&ly*o]M̹Qh2/woQoPSJŪ[lDY&`Riڊ0z۶ܫ/PK! ѐ'theme/theme/_rels/themeManager.xml.relsM 0wooӺ&݈Э5 6?$Q ,.aic21h:qm@RN;d`o7gK(M&$R(.1r'JЊT8V"AȻHu}|$b{P8g/]QAsم(#L[PK-![Content_Types].xmlPK-!֧6 0_rels/.relsPK-!kytheme/theme/themeManager.xmlPK-!wG theme/theme/theme1.xmlPK-! ѐ'Q theme/theme/_rels/themeManager.xml.relsPK]L s 55BBBE   6=?E!8@0(  B S  ?%6{HQfw '83DS3333333333333@BNv{@BNv{'&Mmp. %*Mr2dQ7; BcH7TAVYs,[QycRdgxomu$w./|fL0([sFu7bR%nO()U&d@0@UnknownG.[x Times New Roman5Symbol3& .[x Arial3. AptosC. Aptos DisplayA$BCambria Math"1hx'BgFDFD!03q@P ?72!xx e  Prepare 0jhousele Jon Houseley Oh+'0|  8 D P\dlt Prepare 0 jhouseleNormalJon Houseley5Microsoft Office Word@%x@*@T5$FD ՜.+,0 hp  University of Edinburgh  Prepare 0 Title  !"#$%'()*+,-0Root Entry FI-21Table;WordDocument.SummaryInformation(DocumentSummaryInformation8&CompObjr  F Microsoft Word 97-2003 Document MSWordDocWord.Document.89q