ࡱ> Y[Xi #bjbjzz .ZjjC$ $ 8LO(   ???35555558!5?????5  4J?*  3?3 5Ai^`0|"|"8|"????????55g???????|"?????????$ Y }: PAGE gels Polyacrylamide gels are used for small nucleic acid fragments. They can be either denaturing for RNA and single stranded DNA, or non-denaturing for double stranded DNA. Running a denaturing PAGE gel The trick to getting a nice denaturing 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 800ml 1xTBE to ~55( (3 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)5130356106298762610551215288 Running a non-denaturing PAGE gel These instructions are for a 1mm wide, 7cm acrylamide minigel Make up a 10ml stock of PAGE solution of the appropriate percentage: 1ml 10x TBE The required amount of 29:1 acrylamide from 30% stock Water to 10ml 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 normal 6x loading dye Pull the comb, clean wells with syringe and immediately load samples Run at 120V for ~45 minutes (7cm minigel) % acrylamideXylene cyanol (nt)Bromophenol blue (nt)526065816045127020156015 Staining (denaturing or non-denaturing) Remove gel from glass plates and place in 50 ml 0.5x TBE (or 1x) with 5(l SYBR Gold (you can also use ethidium), stain in shaker for 5 minutes then dispose to ethidium waste and destain gel for 5 minutes with 1x TBE. Transfer and fixation For small fragments (maybe 100nt or less), use EDC fixation, though beware that this relies on a free terminal phosphate group on the DNA/RNA being blotted. For larger DNA/RNA fragments, UV fixation is effective and simpler. For UV fixation Transfer to Hybond N+ using Invitrogen NOVEX transfer system, according to manufacturers instructions, 1-2h at 30V. Dismantle the blot, check by UV that the DNA/RNA has transferred. Rinse the membrane a few times with water. Dispose of the gel and leave the membrane on the 3MM paper, fix in the Stratalinker on Auto. For chemical fixation Transfer to Hybond N (not N+) using Invitrogen NOVEX transfer system, according to manufacturers instructions, 1-2h at 30V. Before the blot is over, pre-heat a hyb-oven to 60( Make the fixation solution (enough for 2 membranes): In a fume hood, add 61l 1-methylimidazole to 6ml water, and add 6l conc. HCl, mix. The pH should be 8.0. Add 0.19g EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide], shake to dissolve. EDC is stored at -20( Dismantle the blot, check by UV that the DNA/RNA has transferred. Rinse the membrane a few times with water. Place a piece of 3MM paper just larger than the membrane on a sheet of Saran Wrap large enough to wrap it, and soak the 3MM paper with the fixation solution Lie the membrane RNA side up on the 3MM paper, wrap in the Saran wrap, wrap in a second layer of Saran and bake at 60( for 1hr In a fume hood, un-wrap the membrane and dispose of the saran and 3MM paper. Wash the membrane a few times with water to remove residual fixative. 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 Add 1ml EtOH, 1(l glycogen and precipitate, wash with 70% ethanol, re-suspend in desired volume 0.1x TE (or whatever) Running large denaturing PAGE gels This is for our system with 20x20cm glass plates, 1mm spacers, but beware that systems will differ! You may need more gel and the voltages may be different 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, put on shaker 5min Pour the staining solution in the ethidium waste, destain 5 min with 1x TBE and image Solutions 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      FILENAME PAGE gels 4.0 Houseley lab  PAGE 1   5HZl Q R S p      % 0 k { ~  ǿǡǡǥǡǡǡhq6 jhJ!hJ!hcDhHeh\-h 6 h 6 hHe6h\-hHe6h2mhC_hC_5\h*lhC_5\ h*l5\h.U hC_hC_hC_hC_5CJ(\aJ(;  Q R  0 k ~  , - C ` { gdq6gdHegdC_$a$gd.U    " + , - 4 6 9 C F G X _ ` c d k z { | 67WXYZjk|}678AEPsh#rh_hn]hRhl"hC_ jh2mhHehz*}h1.hBYh.U jmh.Uh eh2mhJ!h\-hq6C{ | 67YZ67 $$Ifa$gdCgd2m%&)*+,Ihijov 8<=>@IJKRTWadh*l h*l6h\-h*l6hC_h*l5\h*lh*l5\ h*l5\h hRhChR5K $$Ifa$gdCgkd$$IflF     t6    44 laytC $$Ifa$gdCgkdP$$IflF     t6    44 laytC $$Ifa$gdCgkd$$IflF     t6    44 laytC $$Ifa$gdCgkd$$IflF     t6    44 laytC $$Ifa$gdCgkd@$$IflF     t6    44 laytC*+ijJKa~gd*lgkd$$IflF     t6    44 laytCdev}~ "#CXYZj     !#$&')*+,-./08@QW h 5hC_hC_5h hCh*l5 h*l5h*l jmh*lS"#YZgkd$$IflF     t6    44 layt\S $$Ifa$gd\Sgd*l   gkd0$$IflF     t6    44 layt\S $$Ifa$gd\S  $$Ifa$gd\Sgkd$$IflF     t6    44 layt\S !$'* $$Ifa$gd\Sgkd$$IflF     t6    44 layt\S*+,-. $$Ifa$gd\Sgkd $$IflF     t6    44 layt\S./0XY23IJ+,<= !gd*lgkdp$$IflF     t6    44 layt\SWXY23HIJN_egrsy*+,;<=FS !8h~տͿʹʹͭ h*l6] h*lh*lhRhq6h*lh*l6]h*lhC_h#rhC_h#r5 h*l5 jmhC_ hC_\ hC_5hC_hC_\D!~HI~RT_`stuUVgd*lFGHI}~<PQRST}^_`rstuGUVtu۹۵hs` jmh#rhA@n jh#rhghz*}hHehbeh#rhq6h#r5hR jh*lh*l h*lh*lh*lFVtuNOPstVy@Acdgd %&1;@MNOP^irstVy@Acd9no   1 2 ] _ !!! jmh hBs jh h h 6 h 6h h 5\]hRh#rhs`h J9no  ] _ !!2!3!!!!!!!!gd !2!3!s!t!!!!!!!!!!!!!!!!!!!""9"R"_"n""""""""""""""""""""""""""""""""###ļhHehC_hn]hC_mHnHuh\-jh\-Uh\Sjh\SU h h jh h 5\h h3OJQJ^Jh3h3h36>!!!"9"R"_"n""""""""""""""######gd ## # # # ##### h h h h\-h,a90JmHnHu h\-0Jjh\-0JU ,1h. 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