ࡱ> 352 Wbjbj 0"I jjjjj~~~8,~x,3j3jjHJjjf^0j$33 0:    FISH Probe Labelling using Bioprime Labeling Kit 1. Dissolve ~100 ng DNA in 19( of TE buffer. - 40 ng in 7.2ul in TE 2. On ice, add 20( 2.5X Random Primers Solution. - 8ul random primers 3. Denature by heating for 5 mins. Using a heat block set at 100C. - use PCR block for small volumes 4. Immediately cool on ice. 5. Add 5( of homemade dNTP Mixture (See below). - 2ul dNTP 6. Add 5( of Cy3-dCTP or FITC-dUTP or Cy5-dCTP. - 2ul fluorescent analog 7. Add 2( of Klenow fragment. - 0.8ul Klenow 8. Incubate at 37C O/N. - use PCR block for small volumes 9. Add 5( of Stop Buffer. - 2ul stop buffer 10. Purify probe using a G50 Sephadex Column. - add 30ul of TE before spinning 11. Add 10( of yeast tRNA (20 mg/mL stock). 12. Add 3M Na-Acetate to a final conc. of 0.3M. 13. Add 2.5 volumes of 100% EtOH. 14. Spin down, 20 at 40C. 15. Resuspend pellet in 360( of ddH2O and add 40( of 3M Na-Acetate, add 1 mL of 100% EtOH. - 144ul H20, 16ul Na-Ac, R/S and mix well - 400ul EtOH 16. Store all labeled probes at -200C. Good indefinitely in EtOH. Precipitated probes (see below) only good for about a month. Cy3-dCTP or Cy5-dCTP Homemade dNTP Mix FITC-dUTP Homemade dNTP Mix 2mM dATP 2mM dATP 2mM dGTP 2mM dGTP 2mM dTTP 1mM dTTP 1mM dCTP 2mM dCTP Cy3 dCTP GE 25nmol PA53021 Cy5-dCTP GE 25nmol PA55021 FITC-dUTP Roche 25nmol 11373242910 FISH PROBE PRECIPITATION (DNA Probes) 1. Aliquot 100( of each probe in Ethanol. (50ul of each) 2. Add 300 (g of tRNA (15 (L of 20 mg/mL stock). 3. Add 15 (g of mouse COT-1 DNA (15 (L of 1 mg/mL) Heat at 95C for 5 mins, then place on ice for 2 mins prior to use 4. Add 150 (g of sheared boiled Salmon Sperm DNA (15 (L of 10 mg/mL). Heat at 95C for 5 mins, then place on ice for 2 mins prior to use 5. Add 10( 3M NaAcetate. 6. Add 45 (L of ddH2O. (vortex) 7. Add 250 (L of 100% EtOH. 8. Spin to precipitate probe mix. (10 minutes at top speed in cold room) 9. Wash precipitated probe with 70% EtOH (add and vortex, spin for a minute or two, aspirate off EtOH using pipettor so that you dont suck up the pellet). 10. Wash precipitated probe with 100% EtOH. 11. Dry in speed vacuum without heat for 5 min. (alternatively, air dry). Cannot have even a trace of EtOH in probe. 12. Resuspend in 50( of 100% Formamide. (let sit at RT for 5-10 minutes) 13. Denature at 800C for 10 minutes. (after 5 minutes tap tube gently to help dissolve pellet and then put back on heat block another 5 minutes) 14. Quick cool on ice. 15. Add 50( 2X hyb. solution. 16. Preanneal at 370C for 1-1.5 hours. 18. Use 8-10( of probe per coverslip. 19. Store remaining probe at 200C. (Do not preanneal before use.) 2X Hybridization Mix 1 Part Sigma ddH2O 1 Part 20X SSC 1 Parts 10mg/mL BSA (NEB 100X BSA commonly used for restriction enzyme digests) 2 Parts 50% Dextran Sulfate     23PQM N R Z @ A O P o p w x j s t '  9:ɷɭ jmhohWho5>*h_#ho5>*h_#ho5 hoH* hoH* ho5 hhohho5hho5>* jlhoho ho5>* hp5>*=23`xy ' ( D E u  7 8 R e $a$gdo$a$gdoe f   7 8 S T , i j  $ &^`a$gdo$ &^`a$gdo$a$gdo  % & ' B ] HI12RSgdo$a$gdogdo$ &^`a$gdo:GH!"<=EF^_pqu?@8;XY"#CDHIJLMOPRSVWٽhpjhpU hhoh0hoH* h0ho hoH*hoOJQJo(hTOJQJ jmhoOJQJhoOJQJhoH*OJQJ jlho jmhohohT5SpqWXEF67_`-gdogdo-IKLNOQRTUVWgdTgdo .:po/ =!"#$% 666666666vvvvvvvvv666666>6666666666666666666666666666666666666666666666666hH6666666666666666666666666666666666666666666666666666666666666666666666666662 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~_HmH nH sH tH @`@ NormalCJ_HaJmH sH tH J@J A Heading 1$@&5>*OJPJQJaJDA`D Default Paragraph FontRiR  Table Normal4 l4a (k (No List JJ AHeading 1 Char5>*CJOJPJQJB>@B ATitle$a$5>*OJPJQJaJBB A Title Char5>*CJOJPJQJ4@"4 T0Header  !6/16 T0 Header CharCJaJ4 @B4 T0Footer  !6/Q6 T0 Footer CharCJaJPK!pO[Content_Types].xmlj0Eжr(΢]yl#!MB;.n̨̽\A1&ҫ QWKvUbOX#&1`RT9<l#$>r `С-;c=1g'}ʅ$I1Ê9cY<;*v7'aE\h>=,*8;*4?±ԉoAߤ>82*<")QHxK |]Zz)ӁMSm@\&>!7;ɱʋ3װ1OC5VD Xa?p S4[NS28;Y[꫙,T1|n;+/ʕj\\,E:! t4.T̡ e1 }; [z^pl@ok0e g@GGHPXNT,مde|*YdT\Y䀰+(T7$ow2缂#G֛ʥ?q NK-/M,WgxFV/FQⷶO&ecx\QLW@H!+{[|{!KAi `cm2iU|Y+ ި [[vxrNE3pmR =Y04,!&0+WC܃@oOS2'Sٮ05$ɤ]pm3Ft GɄ-!y"ӉV . `עv,O.%вKasSƭvMz`3{9+e@eՔLy7W_XtlPK! ѐ'theme/theme/_rels/themeManager.xml.relsM 0wooӺ&݈Э5 6?$Q ,.aic21h:qm@RN;d`o7gK(M&$R(.1r'JЊT8V"AȻHu}|$b{P8g/]QAsم(#L[PK-!pO[Content_Types].xmlPK-!֧6 -_rels/.relsPK-!kytheme/theme/themeManager.xmlPK-!!Z!theme/theme/theme1.xmlPK-! ѐ'( theme/theme/_rels/themeManager.xml.relsPK]# W " :W e  S-W 8@0(   B S  ? $[_|15Xa%)  $+/bf\`z~ &/im '/~    I K L N O Q R T X  8Q,hJP% ( l q  I K L N O Q R T X ::::::::::poTI K @H H zH H W x@UnknownG*Ax Times New Roman5Symbol3 *Cx ArialK2Times New Roman3TimesA$BCambria Math qhY&'J'   24D D K#Q?O^2 ,1FISH Probe Labelling using Bioprime Labelling KitSundeep KalantryMauro Calabrese Oh+'0 4@ d p | '4FISH Probe Labelling using Bioprime Labelling KitSundeep Kalantry Normal.dotmMauro Calabrese13Microsoft Macintosh Word@40@s\@@Xf  ՜.+,0< hp  '(Univ. of North Carolina, Chapel HillD  2FISH Probe Labelling using Bioprime Labelling Kit Title  !#$%&'()+,-./014Root Entry Ff61TableWordDocument0"SummaryInformation("DocumentSummaryInformation8*CompObj` F Microsoft Word 97-2004 DocumentNB6WWord.Document.8