ࡱ> Z\Yc hbjbj .8ObObQ  8$\0XX([[[KMMMMMM$ q[[[[[qSSS[KS[KSSS/1jS70S@!@!SS@!g[[S[[[[[qq[[[[[[[@![[[[[[[[[ B R: Denaturing poly-acrylamide gel The trick to getting a nice PAGE gel is that the gel should be hot and the heat distributed uniformly. This is exactly the opposite of what you want in a protein gel, and protein gel tanks are designed to dissipate heat. We routinely use old school Biorad minigel tanks that have a Perspex block held against the gel (that is what this protocol is written for). For larger gel tanks, or other styles, a ~1mm sheet of aluminium sandwiched against the outside of the gel works well to spread the heat. Make sure that this is high enough not to dip in the buffer and low enough so that you can see your samples as you load them. These instructions are for a 1mm wide, 7cm acrylamide minigel: See later for large gel instructions Make up a 10ml stock of PAGE solution of the appropriate percentage: Weigh 4.8g urea into a 15ml tube Add 1ml 10x TBE Add the required amount of 29:1 acrylamide from 30% stock Add water to 10ml Heat gently and swirl (use a 55( water bath) until urea dissolves Top up to 10ml exactly with water Clean glass plates with water then ethanol, assemble with 1mm spacers Add to 10ml PAGE mix: 50(l 10% APS, invert to mix 10(l TEMED, invert to mix Pour immediately and insert the comb (make sure this is 1mm) Allow to set for 30min, then assemble the apparatus but leave the comb in Meanwhile, mix samples 1:1 with PAGE loading dye Denature samples 5 minutes at 95( Meanwhile, warm 500ml 1xTBE to ~55( (2 min in our microwave) and pour in tank Snap chill samples on ice, spin briefly and keep on ice until loading Pull the comb, clean wells with syringe and immediately load samples Run at 300V (7cm minigel) (Really! this keeps the gel hot), dye migration is given in the table below % acrylamideXylene cyanol (nt)Bromophenol blue (nt)5130356106298762610551220288 Blotting: Wash gel 2x 5 min with 0.5x TBE, first wash containing SYBR if image is needed. Transfer to Hybond N+ using Invitrogen NOVEX transfer system, according to manufacturers instructions. Band extraction: Locate band by SYBR gold staining or autoradiography and excise with a blade Wipe the handle of a plastic spreader with RNAseZap (Ambion) Mash up gel splice in 1.5ml tube using handle of plastic spreader Add 0.5ml PAGE elution buffer Incubate at 37( with agitation for 1-3hrs Spin buffer through Spin-X column to remove gel pieces Phenol chloroform extract Add 1ml EtOH, 1(l glycogen and precipitate, wash, re-suspend 2x PAGE loading dye: 95% formamide 0.025% bromophenol blue 0.025% xylene cyanol 5mM EDTA 0.025% SDS store long term at -20(, up to 1 month at RT PAGE elution buffer: 0.5M NaOAc 1mM EDTA 0.1% SDS For large PAGE gel (20x20cm glass plates, 1mm spacers): Systems will differ! Make up a 35ml stock of PAGE solution of the appropriate percentage: Weigh 16.8g urea into a 50ml tube Add 3.5ml 10x TBE Add the required amount of 29:1 acrylamide from 30% stock 17.5ml for a 15% gel (my normal) Add water to 35ml Heat gently and swirl (use a 55( water bath) until urea dissolves Top up to 35ml exactly with water Clean glass plates with water then ethanol, assemble with 1mm spacers Add to 35ml PAGE mix: 175(l 10% APS, invert to mix 35(l TEMED, invert to mix Pour immediately and insert the comb (make sure this is 1mm) DON'T INSERT COMB ALL THE WAY! about 1cm is right Allow to set for 30min, then assemble the apparatus but leave the comb in Meanwhile, mix samples 1:1 with PAGE loading dye Denature samples 5 minutes at 95( Meanwhile, warm 700ml 1xTBE to ~55( (3 min in our microwave) and pour in tank Snap chill samples on ice and keep on ice until loading Pull the comb, clean wells with syringe and immediately load samples Run at 500V until dyes have migrated the required distance Staining with SYBR Gold Put gel directly into plastic box with 100ml 1xTBE containing 10l SYBR Gold Cover with aluminium foil (I keep a large piece for this), put on shaker 15min Pour the staining solution in the ethidium waste, rinse the gel with water and image      FILENAME Denaturing PAGE gel.doc 3.1 Houseley lab  PAGE 1       * + > F G V _ ` f   ' , 2 [ _ ` a c l u w z ¾ܶܶܺܶܶܶܧܶh1.hBY jmh.Uh eh\-hq6 jhJ!hJ!hcDhHeh\-h 6 h 6 hHe6h\-hHe6h2mh.Uh.Uh{5CJ(\aJ(h.Uh.U5CJ(\aJ(8  > ` q   % & m n E F gdq6gdHe$a$gd.U  . 2BYyKUbh!16:Canp 579:sٽ͹͹͹͵ٵٽ͹ͱͪͦhA@n jh#rhghq6hbehq6h#r5hChR5h#rh_hn]hRhl" jh2mhHeh.Uh\-h2mhJ!hz*}@F w x 12wx $$Ifa$gdCgd2m # $$Ifa$gdCgkd$$IflF     t6    44 laytC#$&*- $$Ifa$gdCgkdP$$IflF     t6    44 laytC-.036 $$Ifa$gdCgkd$$IflF     t6    44 laytC67:=@ $$Ifa$gdCgkd$$IflF     t6    44 laytC@ADGI $$Ifa$gdCgkd@$$IflF     t6    44 laytCIJKUV!"opgkd$$IflF     t6    44 laytC;<st(5DuvwDgzgd ^_w    ab "#-.A»»ҳ}hn]mHnHuh\-jh\-Uh,a9jh,a9U h h3h3OJQJ^Jh3h3h36 jmh hBs jh h 6h h$Zh jh_h+hh_hRhs`h#r jmh#r0z./QR'\]KMgd  !no!"XYefghgd AEFHJKSXZ[abcdfgh޿ h h3h,a9h,a90JmHnHu h\-0Jjh\-0JUh\-hHeh hn]jh\-Uh_mHnHu,1h. 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[ @VerdanaA$BCambria Math"1h8GJ f f !42q HP ? 2!xxqp Denaturing poly-acrylamide gelHouseley Jon Houseley Oh+'05 ( H T ` lx Denaturing poly-acrylamide gel HouseleyNormalJon Houseley6Microsoft Office Word@@֫@x廅@f Gh3 Rt6   d.@Times New Roman--- (2 =xcDenaturing PAGE gel   2 =c.doc  2 = c c  2 =c3 2 =c.1  2 =c  2 =PcHouseley   2 =c  2 =clab  2 =c  c''  2 /xc   2 /c1  2 /c  c''@Times New Roman--- "2 ycDenaturing poly       2 yc-   2 ycacrylamide gel     2 yBc  ---  2 xc   2 xPcThe trick to getting a nice PAGE gel is that the gel should be hot and the heat     2 xWcdistributed uniformly. This is exactly the opposite of what you want in a protein gel,         a2 x9cand protein gel tanks are designed to dissipate heat. We  12 croutinely use old school    2 xWcBiorad minigel tanks that have a Perspex block held against the gel (that is what this        2 xQcprotocol is written for). For larger gel tanks, or other styles, a ~1mm sheet of       j2 x?caluminium sandwiched against the outside of the gel works well        )2 cto spread the heat.   2 xWcMake sure that this is high enough not to dip in the buffer and low enough so that you     D2 x&ccan see your samples as you load them.    2 qc    2 2xc  @Times New Roman---  2 ExcT  72 Echese instructions are for a 1 ;2 E5 cmm wide, 7cm acrylamide minigel:       2 Ec    2 Wxc 0 A2 W$cSee later for large gel instructions  2 Wc  ---  2 ixc   2 |xcMake  2 |cup  2 |c  2 |ca 10 .2 |cml stock of PAGE soluti    2 |hco   2 |qcn  2 |xc  2 | cof the appr  2 |co &2 |cpriate percentage:  2 |<c    2 xc 0 2 cWeigh 4  2 c. "2 c8g urea into a  2 > c15ml tube   2 }c    2 xc 0 2 cAdd 1  2  cml 10x TBE    2 c    2 xc 0 a2 9cAdd the required amount of 29:1 acrylamide from 30% stock        2 /c    2 xc 0 "2 cAdd water to 10   2 cml   2 c    2 xc  @Symbol--------- :2 xcHeat gently and swirl (use a 55   ---  2 ?c---  2 Fc  :2 Jcwater bath) until urea dissolve    2 cs  2  c    2 xc   2 x cTop up to 10  +2 cml exactly with water     2 Tc    2 #xc    2 5xcC  2 5clean  2 5cglass  I2 5)cplates with water then ethanol, assemble     2 5cwith   2 5c   2 5c1 2 5cmm   2 5 c  2 5cspacers   2 5Ec    2 Gxc   2 ZxcAdd to  2 Zc10 2 Zcml  2 Z cPAGE mix:    2 Zc  ------  2 nxc 0 2 nc50---  2 ncm --- %2 ncl 10% APS, invert    2 n1c  2 n:cto mix   2 n_c  ---  2 xc 0 2 c10---  2 cm --- 2 cl TEMED   "2 c, invert to mix   2 Xc    2 xc   2 xcPour   2 c  2  cimmediately    2 c  (2 cand insert the comb   2 kc  )2 pc(make sure this is 1  2 cmm)   2 c    2 xc   2 xcAllow    2 c  ,2 cto set for 30min, then   2 +c  ,2 1cassemble the apparatus   2 c  +2 cbut leave the comb in   2 Pc    2 xc   S2 x0cMeanwhile, mix samples 1:1 with PAGE loading dye        2 c    2 xc  --- ;2 x cDenature samples 5 minutes at 95    ---  2 Pc---  2 Wc    2 (xc  --- >2 <x"cMeanwhile, warm 500ml 1xTBE to ~55      ---  2 <yc---  2 <c  I2 <)c(2 min in our microwave) and pour in tank      2 <c    2 Nxc   2 N|c   2 `x cSnap chill   2 `csamples   2 `con  2 `c  R2 `/cice, spin briefly and keep on ice until loading    2 `$c    2 sxc   #2 xcPull the comb, c   .2 clean wells with syringe    2 oc  52 xcand immediately load samples      2 7c    2 xc    2 xcR  2  cun at 300 2 cV (7  2  ccm minigel)    2 $c  2 - c(Really!     2 ec  2 mc  .2 qcthis keeps the gel hot) 2  c, dye migrat  2 G cion is given     &2 xcin the table below   2 c   @Times New Roman--@Times New Roman--- "System5$ w--ccbbaaK ՜.+,0 hp  Edinburgh Univeristy Denaturing poly-acrylamide gel Title  !"#$&'()*+,-./012345789:;<=>?@ABCDEFGHIJKLMNOPRSTUVWX[Root Entry F2]Data 1Table%@!WordDocument.8SummaryInformation(685DocumentSummaryInformation8QCompObjr  F Microsoft Word 97-2003 Document MSWordDocWord.Document.89q