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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 ISN’T! 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 100µl reaction, add 100µl 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 1µl of glycogen, 10% volume 3M NaOAc and 2-2.5 volumes 100% ethanol, vortex well Ie: for 100µl add 1µl glycogen, 10µl 3M NaOAc and 250µl 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 Æ Ç È    $ % 9 : F _ û ü  ,RSVlno–˜°½¾Çþÿ@A�‚ƒœ¥³ÂÕÖÙÚþÿöíåÞÞÞÔÞÞÍÞÞÞÂÞÞÞÞ»´»´»ÞÞ­ÞÞÞÞ­Þ­Þ­ÞÞÞ­»´»©»¥»»´Þ´´¥¥´´´©´¥´´´¥´´hU&Ëh¶%« hU&ËhœO· hU&ËhU&Ë hU&ËhÈÅhU&Ëh»AVB*phÿ hU&Ëh™)ÇhU&Ëh»AV5�\� hU&Ëh»AVhMh™)Ç5�hM5�CJ(aJ(hÌ[—5�CJ(aJ(A/0lm¨ © e f Æ Ç È  9 : û ü RS¾ÿA‚ƒÕÖþÿ÷òððððððððððððððððððððððððððgd™)Ç$a$gd™)Çÿ9:vw{‰Š‹Œ�Ž��‘’“”•–—¡¢ª«¹½¾¿ÀÁÇÈÉÊËÌÍÎùùùùõõùêâÞÞâÞÞâÞÞâÞÞÖÒÖÉÖÒÅÒÒÒ»µ»ª»ÒÒ¦êhó7›hñ0JmHnHu h(¹0Jjh(¹0JUh€%h(¹mHnHuh(¹jh(¹Uh jh UhU&ËhU&ËOJQJhU&Ë hU&ËhU&Ë'ÿ9:w‰ŠŒ���’“•–¾¿ËÌÍÎýýýôýýýýýýýýýòýðýýý„Ð`„ÐgdU&Ë,1�h°‚. °ÆA!°"°#� $� %°°Ä°Ä �Äx2ÀÐàð 0@P`p€�ÀÐàð2(Øè 0@P`p€�ÀÐàð 0@P`p€�ÀÐàð 0@P`p€�ÀÐàð 0@P`p€�ÀÐàð 0@P`p€�ÀÐàð 0@P`p€�8XøV~� °ÀЀàð 0@àð 0@àð 0@àð 0@àð 0@àð 0@àð 0@àð 0@àð 0@àð 0@àð 0@àð 0@àð 0@àð 0@66666_HmH nH sH tH @`ñÿ@ NormalCJ_HaJmH sH tH DA`òÿ¡D Default Paragraph FontRióÿ³R  Table Normalö4Ö l4Öaö (k ôÿÁ(No List 4@ò4 Mr2Header  Æ9r 4 @4 Mr2Footer  Æ9r .)@¢. 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