邢唷��>� FI��E��欹�c ��bjbj*�*� .4H許bH許b��D�� :(b ��8�44�f6L(ttt��$k �!#��tt4��t�t��t��衴R佪'��Lp�0�#�0�#�#�,D��#�� B: PFGE gels PFGE gels are almost invariably run using genomic DNA embedded in agarose � see separate protocol for embedding yeast and mammalian cells. Here I simply list a few of the common systems that we use for running the gels. It is really hard to optimise the run parameters, so we generally look for an existing PFGE programme with separation in the appropriate range (look at NEB and BioRad sites which give plenty of programs designed to separate their different ladders). The only tricky thing about these gels is getting the agarose plugs into the wells, and everyone makes a mess of this the first few times. Otherwise these are just gels that run for a very long time. Take care of them though, and make the buffer fresh every time for best results. We use BioRad Pulsed Field certified agarose (162-0137), but suspect that normal agarose would be just as good. However, we have learnt to avoid BioRad MegaBase agarose (which we previously recommended) � good samples often fail to resolve in this agarose for reasons that we have not been able to identify. Digesting DNA in agarose plugs (optional) NEB maintains a list of which enzymes work for digestion in agarose and which do not HYPERLINK "https://international.neb.com/tools-and-resources/usage-guidelines/digestion-of-agarose-embedded-dna-info-for-specific-enzymes" https://international.neb.com/tools-and-resources/usage-guidelines/digestion-of-agarose-embedded-dna-info-for-specific-enzymes Cut plug in half and place in a 2ml tube (always 2ml tubes!) with 100祃 1x restriction buffer (normally CutSmart), incubate 15 min at room temperature Replace the buffer with 100祃 fresh buffer for another 15 min at room temperature Replace the buffer with 100祃 fresh buffer containing 10-20U restriction enzyme, incubate the plugs 4 hours - overnight at 37� Equilibrate plugs 15 min in 1ml running buffer before loading onto gel. Running, staining and blotting a PFGE gel These conditions are for yeast chromosome XII (1 � 4Mb resolution) but the steps are always the same Make 2.5L fresh 1x TBE, stir until fully dissolved Microwave 120ml 1x TBE with 0.8% agarose until dissolved For high molecular weight undigested yeast chromosomes use Biorad PFGE agarose instead (162-0137). For smaller fragments (up to 100kb or so), we just use normal multipurpose agarose (currently from Melford MB1200) Stir until cooled to 55( Meanwhile set up gel caster and use a spirit level to make sure it is flat Put the remaining 1x TBE in the tank, start pump, and set chiller unit to 14( Pour gel and let it set for >30min Remove comb and fill wells with running buffer Remove plug from wash buffer with tweezers Put in Petri dish and cut in half (keep one half in wash buffer at 4() Push � plug into well with tweezers Ensure there are no air bubbles between plug and front of well Remove gel caster edges, but leave gel on the black tray (don抰 dislodge it!) Put gel and tray in the fixture in the centre of the tank Ensure that the wells are at the right end Set the conditions and start the gel: Voltage: 3V/cm Run time: 68 hours Switch time: 300-900s Staining When finished, move gel into 200ml water with 3(l ethidium bromide, Wash the tank with 2.5L water After 30min, rinse the gel with water twice and take a picture Blotting Depurinate in fresh 0.25N HCl for 30 minutes (not more, not less!!) Denature in 0.5N NaOH for 30 minutes Neutralise twice for 15min with 0.5M Tris pH7.5, 1.5M NaCl Transfer to HyBond N+ membrane in 6xSSC PFGE for standard yeast chromosome (200kb � 1.5Mb resolution) Gel percentage: 1.0% agarose Running buffer: 0.5x TBE Switch time: 60-120s Run time: 24 hours Voltage: 6V/cm PFGE for meiotic yeast chromosomes (40 - 400kb resolution) Gel percentage: 1.3% agarose Running buffer: 0.5x TBE Switch time: 15-25s Run time: 24 hours Voltage: 6V/cm The following conditions are from Stults et al PMC2134781, they work in our hands PFGE for human 5S rDNA (50 � 500kb resolution) Digest plugs with EcoRV-HF Gel: 1% agarose 0.5x TBE (fresh) 14癈 6V/cm 120� included angle 3-90s switch 24 hour run time PFGE for human or mouse 45S rDNA (500kb � 6Mb resolution) Digest plugs with EcoRV-HF (human) or PmeI (mouse) Gel: 0.8% agarose 1x TAE (fresh) 14癈 2V/cm 106� included angle 300-2400s switch 92 hour run time Solutions 1x TBE (2.5L): 27g Tris 13.75g Boric acid 10ml 0.5M EDTA pH8 (do not substitute with solid) 0.5x TBE (2.5L): 13.5g Tris 6.9g Boric acid 5ml 0.5M EDTA pH8 (do not substitute with solid) For enzymes that require 50� incubation, increase volume to 200祃, this is ok for overnight Using TBE diluted from even fairly new 10x stocks has a major negative impact on gel quality Use a fresh Petri dish each time you load a gel, or wash it well See our genomic southern protocol for detailed method, but note the differences in treatment here Concentrated HCl (37% aqueous solution) is 12N You need a CHEF-DRIII for this FILENAME PFGE gels v3.0 Houseley lab PAGE 4 m � +3��12P[\��? @ A � � Z~��*+,x��鲰殄徨阱徨徨徨治晌至侄镰林ブ炛ブ稚埳亅woh]a�� 耥殄坶碜禹酉尤幽斫砉礤黹瑰楣槟诞鬼屙屙犿湑滍瑰瑰瑰瑰瑰 j梆h?5h?5jh窧�0JUh�/ j梆h=?�h�h=?� j梆h岻h$gh�9 h�9 h�h�9 h�'jh�9 0JUh窧�h鈄�h岻h.Rh盜vh.Rh?xNOhi��'(W��23��勑`勑gd窧�勑^勑gd�9 13@IJSTcgw}�� !"(0GH�� 祠蔹滂天豇蠕染萨壶轰轰轰轰淡槰h窧�h@b>=?� Footnote TextCJaJ@&`�q@=?�Footnote ReferenceH*6U`��6�� Hyperlink>*叠*辫丑肠�<��/��<�.RFootnote Text CharPK!檗�[Content_Types].xml瑧薔�0E鱄鼉�-J湶@%閭菐洽|廊�$韶钵UL襎B� l,�3鳛;鉹�得槣B+$�G]ミ7O侪V墎�c:菜�E3v@釶莮�憇� |煓w�<�<爔k猴ll5� 玈Z埣2O,辜帣Q績� €‰壪�5y�;y=卐E颎翼帊|\P疼鞭M7�$|n]�枌蓢]4忥`窾6涀G冼(垲�^斗菹颻琖��-翻関]m蓿瓄�  飍祡缧暺}攙旯缦r�鍧竔倲崜0�� D3劂梋閐�3譙钐嚟�耳杧:忲�8}d-楀鉯*壵x上轻qCよZ}�柣WlшqyI@赹剟6橧!Q_� ﹪s殔刏賩a汛癶H魉Tm�jyV`垆纅�榾惫�<箣犇鼁YpL8�*аx侏S&机;EUC�8⒊eE�<В悟hg贇!燴H睩x0� VM螽憆刳u�6拺覦s�3 U慮赢b��6�怂5y嵳2臓iz嘜]r汯璠�'鋆炃弦u涎4jj採�K臀F娃盶�晕�$4寨-莓�-�珠`餜�熘��&藑代觥瀈A<吼"xNW﹦� � 袬鞩R賭[鋺萵 8r� i箯K~�*~P(5黕莲z胦Wm忒杮~乖鞹濦caT鲇�.}x E僮5抉&Z緄�2bQ懇/,EE\}�)W�乹埼鉠ミ�6;礏弛�糿h礜贻~7�威�9R`痌糧疩〞儬嗾J拁▄旿郢�=$燮朗Sb酻紇��PK! 褠煻'theme/theme/_rels/themeManager.xml.rels剰M �0匃倃oo雍�&輬协�勪5 6?$Q祉 �,.嘺緳i粭澤c2�1h�:闀q毩m胳嶡RN壻;d癭値o7�g慘(M&$R(.1榬'J摐袏T鶂�8V�"＆A然蠬鱱}狇�|�$絙{�朠�除8塯/]As賲(⑵锑#洩L蔥汉倪��PK-!檗�[Content_Types].xmlPK-!ブх�60_rels/.relsPK-!ky��theme/theme/themeManager.xmlPK-!稝��theme/theme/theme1.xmlPK-! 褠煻'� theme/theme/_rels/themeManager.xml.relsPK]� +<� ��]��c��%4�� 66CCCF� �� @��X�€!7>@F�晙!�晙餈� @��€€€�餒 ��0�( � �饞��0�( � �養 �S�� ?�� PV��+/��QT��m��$+,� � � � ��. ��m��$+,� � � � ��. ��/7iK�J閆B#鷘EI� /O�9 岻琈:l>H"K($g�'>7)iO*�,�/)2鐃4?5�C@#D闟D*GBJ�L.RPuV\,X#YX?Kab瀅cjCl&il<*n�&r�>sPVtm��<��A�=?�MC��@€0000�(@��Unknown��G��.郲x� �Times New Roman5�€Symbol3.��.郲x� �Arial7.��.鄘$� �Calibri5.��.醄`�)�TahomaA��$B�Cambria Math"1�鹦h刔�'璢�'毋'�5 �5 !��亖4��2僸��HP �?��2!xx�姂��Yeast PFGE plug preparationjhouseleJon Houseley� 鄥燆鵒h珣+'迟0�� $0 P\h t€��Yeast PFGE plug preparationjhouseleNormalJon Houseley3Microsoft Office Word@4�0@霩T鹩�@郖弊'�@択�'��5� 胀諟.摋+,D胀諟.摋+,\hp�� University of Edinburgh �Yeast PFGE plug preparationTitle€ 8@_PID_HLINKS�A8u)https://international.neb.com/tools-and-resources/usage-guidelines/digestion-of-agarose-embedded-dna-info-for-specific-enzymes �� !"��$%&'()*+,-./01234��6789:;<��>?@ABCD��GH��K��Root Entry�� F0 b佪'�J@Data ��1Table��#�#WordDocument ��.4SummaryInformation(��5DocumentSummaryInformation8��=MsoDataStore��R佪'�衴R佪'��N��K�U�JH��TW5TDHA==2��R佪'�衴R佪'�Item �� Properties��UCompObj��r�� F Microsoft Word 97-2003 Document MSWordDocWord.Document.8�9瞦