邢唷��>� 24��1��欹�� `bjbj虤虤 * ` ��,�B, ��,n� �� 5��`�!Z醌��^M�0BU,��$��B��: Large RNA Northern Analysis With homemade hyb (from Sharp lab and Bhardwaj and Katiyar-Agarwal et al) The day before, precipitate 10 to 40ug of RNA in EtOH overnight. Pour agarose/formaldehyde gel For 110ml gel: 11ml 10X MOPS 79 ml H2O 20ml 37% formaldehyde 1.1g agarose (EtBr) or stain later with Sybr Gold For 170ml gel 17ml 10x MOPS 122 ml H20 31 37% formaldehyde 1.7g agarose (EtBr) or stain later with Sybr Gold Let gel equilabrate for at least 30min in 1X MOPS buffer R/S RNA in 1x loading buffer RNA should be DNAse treated for best results 1x load: 3.5ul H20 5ul formamide 1.5ul 10x MOPS 2ul 37% Formaldehyde (6% final, like gel) Incubate 5-10 min at 65, chill on ice Add 1/6th volume of loading dye mix and load into gel Run @ 5V/cm with buffer recirculation every 20 minutes Soak 2x for 15 min in H2O to remove formaldeyde Stain and take pic of rRNA bands 28S is 4.7b 18S is 1.8 kb 28S should be twice intensity of 18S Set up transfer in 10X SSC One large wet whatmann for wick Gel, face down Pre-wet hybond N membrane (amersham) 3X wet whatmann, gel/membrane size (make sure no bubbles between sheets!!) 5 cm single fold paper towels, gel size, dry weight Transfer O/N Disassemble transfer unit, rinse membrane in 2X SSC Air dry membrane UV crosslink Pre-wet membrane in 2X SSC Pre-pre hyb at 42 in 2x SSC 0.5% SDS 30 minutes Pre-hyb membrane at 42 for 4 hours to overnight (20ml � save 800ul for probe): 50% formamide 10ml 5x SSC 5ml 0.5% SDS 1ml 5X Denhardts 2ml 5% Dextran sulfate 2ml 150ug/ml Salmon sperm DNA 300ul (boiled!) 150ug/ml yeast tRNA 150ul Vortex Spin 5 minutes at 1000g to remove bubbles Place at 42 C water bath Add to membrane after pre-pre hyb Make probe Amersham/GE ready to go DNA labeling beads Spin purify probe in S-200 column Denature at 95 for 3 min, cool on ice Add to pre-heated 800ul Hyb buffer Add to hyb tube (on side, not directly on membrane) Hybridize overnight @ 42 Rinse with 5x SSC, 0.1% SDS Wash @ 42 in 5X SSC, 0.1% SDS for 10 min, 2x Wash @ 42 in 2X SSC, 0.1% SDS for 10 min, 2x Wash @ 50 C in 0.1X SSC, 0.1% SDS for 20 min, 2x (Wash @ 60 in 0.1X SSC, 0.1% SDS for 20 min, 2x) Wrap membrane in saran wrap and expose on phosphoimager Strip with boiling H20, 0.5% SDS with agitation for 10 minutes, 30 minutes total 6x northern dye 100ul 10x MOPS 200ul glycerol 800ul h20 bromophenol and xylene cyanol 500ml 10X MOPS 41.9g MOPS 4.1 g NaAc H20 to 500 20ml 0.5M EDTA pH to 7 w/ NaOH 1000ml 20X SSC 175g NaCl 88g NaCitrate tribasic, dihydrate H20 to 1000 pH to 7 w/ Hcl 50X Denhardt solution 50mL: 0.5g Ficoll Type 400 0.5g polyvinlypyrrolidone 0.5g BSA Fraction V H20 to 50ml �@ A � � �HIJY��`耥皴褶褡褶褶褚� h(z�5�hy%h(z�h(z�5�>*h$!h(z�H*h�*h(z�h貄wh貄w5�>*gh�� 7 8 F T _ s € � � � � �� &Fgd(z� &Fgd(z�刾^刾gd(z� &F &F勑^勑gd(z� &F &Fgd(z�$a$� � * 3 = K Z � � � � Ijv��c�� &F刾^刾gd(z� &Fgd(z� &F勑^勑gd(z�� B r � � � � � � 29c|��Au�� &Fgd(z�劆^劆gd(z� &Fgd(z� &F��6g��!+IJYdoz��gd(z� d�1$gd(z�1$ &Fgd(z� &F��%?S_`��gd(z� (靶/ 班=!�"�#悹$悹%�靶靶愋��666666666vvvvvvvvv666666>666666666666666666666666666�6666666666�666666666666hH66666666666666666666666666666666666666666666666666666666666666666�66666666662�� 0@P`p€��2(�� 0@P`p€�� 0@P`p€�� 0@P`p€�� 0@P`p€�� 0@P`p€�� 0@P`p€�8X�V~ OJPJQJ_HmH nH sH tH <�`�<�NormalCJ_HmH sH tH DA ��DDefault Paragraph FontVi��V�0Table Normal :V�4�4� la�(k ��( 0No ListV﨩VAF_Title_Running_Head$d�a$PJPK!涜pO�[Content_Types].xml瑧薺�0E鲄䞍卸豶�(ヘ微廬嬼yl嬝#!MB蝼;.�卬姨谨台\艫�1&绌耀夹生芉W橄蚄v疷b�OX�#&疆释1`RⅵT闉9<搇�#ぼ$┐>幚r崫 `沸」-�;c=1g词'縸�虫笔咓颈叠$篒�翱�1脢办9报滨鬰尝�涢贬�隮<�;*v�7'榓瓻\�h>嗟�=青镑,�*彿阗�8辩赌;湷镑�*4髑槿?�奥辩调苍阍塷驳淎撙>曧�8蹿�2�*<")QH裤麆xK┟� 梶ㄐ]奪榸)橛亾MS欯\&>!�7;飞笔�3熥摆淅贰叠鲍缚�1綩楥�5VD翕� 豖靉?p墾勠 S喧4坻曐[驨S叫28;嶻慵[戢櫌�,荇撘骉1|筅n�;�+衡/蕰j�蟎阉\,E:!莒宼�4.T獭e1 �謢�; [鄗^鵳縧�銨�ok侮0e g@礕GHPXNT,賲禿诧e鮸楸*YdT臷橸�鋩熬顏+�(員7�$ow2�茜紓#紾┲浻墒ビ掷?絨迸N澵K椟-�/M,W鏖阦鍗x盕V�/Q吴范朞鯕&渆刀c蛒几\Q滜她LW@H��!�+魝阽{衃|{包!汝K韩A�i `遚m2iU栚|纾Y+�蕤栿� [[v杧煋靣錘缭釫挐3靝m擎R �=Y�04,�!&0�+W魿軆@o習�樫OS2�'S佼0�5嘌$�嗓]pm珠3孎瞭�崕婓G蓜-!鹹へ"�訅V .� 甡幾�,�O.%烫胁Kas楁S球骗弙€稭謟瓔玚姤叢3铵蘽�9+e鰻@�e觇諗錖y硥�7啠W萧_Xtl斴��PK! 褠煻'theme/theme/_rels/themeManager.xml.rels剰M �0匃倃oo雍�&輬协�勪5 6?$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-!涜pO�[Content_Types].xmlPK-!ブх�6-_rels/.relsPK-!ky��theme/theme/themeManager.xmlPK-!!Z!��theme/theme/theme1.xmlPK-! 褠煻'( theme/theme/_rels/themeManager.xml.relsPK]# ` ��` � ��` �8�@��€€€�饞��0�( ��y婚7�zQ�U �養 �S�� ?�*-BJO^��-1<A��=H_c��'FI��%)��69HK��+ 6 B H j n � � � � � � � � � * > b 8E��+ 6 � � � � H P b ::::::<5�簨奬��勑剺��^勑`剺�5o()劆剺��^劆`剺�.刾凩��p^刾`凩�.€凘剺��@^凘`剺�.€�剺��^�`剺�.�勦凩��^勦`凩�.€劙剺��^劙`剺�.€剙剺��€^剙`剺�.�凱凩��P^凱`凩�.<5�� *貄w(z�` b �@€AAzAA` X@��Unknown��G��*郃x� �Times New Roman5�€Symbol3��*郈x� �Arial3�TimesA�Cambria Math A�鹦hT'6�'g峑G I��!��€24[ [ �K#Q��?'��Ui�2,��Large RNA Northern AnalysisMauro CalabreseMauro Calabrese� 鄥燆鵒h珣+'迟0�� $ HT` lx€��'Large RNA Northern AnalysisMauro CalabreseNormal.dotmMauro Calabrese9Microsoft Macintosh Word@霎2 @㑳黔f�@樰�f�@�U醌�� 胀諟.摋+,0hp|�� 'MIT[ Large RNA Northern AnalysisTitle �� "#$%&'(��*+,-./0��3��Root Entry�� F�)Z醌�5€1Table��WordDocument��* SummaryInformation(��!DocumentSummaryInformation8��)CompObj��`�� F Microsoft Word 97-2004 Document��NB6WWord.Document.8