ࡱ> AC@M "bjbj== %<WW6l  , $   L LLL & L LL:Z, @X M > 0Rk!Lk!LStandard Operating Procedure TITLE: MICROARRAY LABELED PROBE HYBRIDIZATION AUTHOR: Modified by Ben Isett, originally obtained from TIGR/Renae Malek (06/24/02) 1. PURPOSE This protocol describes the hybridization of a Cy labeled cDNA probe (mix of Cy3 and Cy5) onto coated slide spotted with oligonucleotides (70mers). 2. SCOPE This procedural format is currently utilized by Duke Microarray Core Facility for hybridization of oligonucleotide arrays 3. MATERIALS 3.1 20X Saline-Sodium Citrate (SSC) (Sigma; Cat # S-6639) 3.2 10% Sodium Dodecyl Sulfate (SDS)(Life Technologies; Cat # 15553-035) 3.3 Bovine Serum Albumin (BSA) 30%,Sigma 3.4 Formamide, redistilled (Life Technologies; Cat # 15515-081) 3.5 Isopropanol (Fisher Scientific; Cat # A451-1) 3.6 Coplin jar (VWR; Cat # 25457-200) 3.7 Human COT1-DNA (Life Technologies; Cat # 15279-011) 3.8 Mouse COT1-DNA (Life Technologies; Cat # 18440-016) 3.9 Poly(A)-DNA, 25U 3.10 Microscope Cover Glass (Fisher Scientific; Cat # 12-545J) 3.11 Hybridization chamber (Corning Costar; Cat #2551) 3.12 1 L .22 mm CA (cellulose acetate) Filter System (Corning; Cat #430517) 3.13 Pressurized air duster (Fellowes; Cat # 99790) 4. PROCEDURE Slide processing: Bake slides for 80 minutes at 80 C UV-Cross link slides with 200mJ of energy Outline array and numbe slide with diamond tip pencil Continue with 4.1 4.1 Prehybridizaton 4.1.1 Prepare prehybridization buffer (5X SSC, 0.1% SDS, 1% BSA) and sterilize by filtration using a CA filter. Preheat at 42oC for ~30 minutes before use. 4.1.2 Place the printed slide(s) which will be used for the hybridization in a Coplin jar containing preheated prehybridization buffer and incubate at 42oC for 30 min-1 hour. 4.1.3 Washing Slides - Fill two Coplin jars with MilliQ water and another with isopropanol. - With forceps carefully grasp slide by the labeled end and vertically dip slide into the first Coplin jar (water) so that the slide is completely submerged. Dip slide four or five times. - Dip the slide again in the water five times but only submerging the slide enough to wash the printed array itself. - Using the same technique dip slide into the second Coplin jar (water). This procedure can be done in glass slide boxes on a nutator mixer. 4.1.4 Drying Slides Spin dry the slide for 1 minute at 1200rpm. - Make sure back of slide is dry as well. - Note the general appearance of the slide. Streaking or mottling on the slide surface indicates further washing is necessary. 4.1.5 Use slides immediately following prehybridization to ensure optimal hybridization efficiency. 4.2 Hybridization 4.2.1 ALWAYS PREPARE FRESH!!! Prepare 1X hybridization buffer (50% formamide, 5X SSC, and 0.1% SDS). 4.2.2 Prepare Poly(A)-DNA by dissolving stock Poly(A)-DNA in a neutral buffer (i.e. 10 mM Tris, pH 7) to a final concentration of 20 mg/mL. 4.2.3 Prepare COT1-DNA (stock conc.1mg/mL) by ethanol precipitation: - Add 2 to 3 volumes of ethanol and 0.1 volumes of 3 M Sodium Acetate (NaOAc) to the stock tube. - Mix well and place on dry ice for 20-30 minutes or in  20oC freezer overnight. - Centrifuge for 20-30 minutes in a cold room microfuge at maximum angular velocity. - Remove supernatant and allow excess ethanol to dry off. - Dissolve precipitated COT1 in a neutral buffer (i.e. 10 mM Tris, pH 7) to the final concentration of 20mg/mL. 4.2.4 Resuspend labeled probe (Cy3/Cy5 probe mixture: see SOP-labeling) in 30 mL of 1X hybridization buffer. Note: Expose Cy labeled probe to light as little as possible during the hybridization process. 4.2.5 To block nonspecific hybridization add: COT1-DNA (20 mg/mL)& & . 1mL Poly(A)-DNA (20mg/mL)& ... 1mL Note: The COT1 DNA is organism specific: add mouse COT1 to labeled mouse probes and human COT1-DNA to labeled human probes. 4.2.6 To denature, heat the probe mixture at 95oC for 2 minutes and snap cool on ice for 30 sec. 4.2.7 Centrifuge the probe mixture at maximum angular velocity for 1 minute. Keep at room temperature and use immediately on baked and blocked array 4.2.8 To Apply Labeled Probe Mixture - Place a prehybridized microarray slide (array side up) in hybridization chamber. - Pipette the labeled probe mixture (~32 mL) to the slide surface near one end of the array print area keeping bubbles to a minimum. - Take a 22mm x 60mm microscope glass coverslip, dust it with compressed air, and grasp one end with forceps. - Holding the coverslip over the array print area, lower the end nearest the pool of cDNA probe until solution wicks to the surface of the coverslip. - Gradually lower the opposite end of the coverslip (held by the forceps) onto the slide. The solution may take a minute or two to wick across the entire length of the slide. - After probe has wicked across the slide carefully adjust the coverslips position with the tip of the forceps so that there is an even margin between the edge of the coverslip and the edge of the slide. - Work any large bubbles toward the edge by gently tapping the coverslip surface; small bubbles will absolve themselves during hybridization. 4.2.9 To the small wells at each end of the chamber add 10 mL of water (20mL total), cover, and seal the chamber. 4.2.10 Incubate in a 42oC water bath for 16-20 hours. To ensure chamber remains level and does not float to the surface place a small weight upon it. Note: Do not flip hybridization chamber upside down during hybridization; this may cause the coverslip to shift from the slide and adversely affect the hybridization. 4.2.11 Wash slides in the following sequence: (wash is performed on nutator mixer) 1xSSC, 0.2%SDS at 42, let sit until cover slip slides off easily (no more than 2-3 minutes) 1xSSC, 0.2%SDS at 42 for 1 minute 0.2xSSC, 0.01%SDS at room temperature for 1 minute 0.2xSSC at room temperature for 2 minutes 0.05xSSC at room temperature for 1 minute Spin dry 2 minutes at 1200rpm 4.2.12 Place slides in a light tight slide box until they can be scanned. $KS>H  SV}MP (:< ^ '(@ "*4rtxz|6CJ]aJCJaJCJOJQJaJCJaJ 56\]6]5CJ\aJ5CJ\aJPKS>?H S}M  " < ` 7$8$H$"` 3 E Y @  > 3 s | 7$8$H$ & F7$8$H$+m"(*0v Gaz7$8$H$"$(*8:\^bdtvzr| LNjlt z !"!q!"""""CJCJaJCJaJCJOJQJaJ5CJ\aJCJaJ1>zr`)24 F`N67$8$H$6dt !!p!q!!!""L"v""""" & F7$8$H$#0P/ =!"#$% i0@0 Normal_HmH sH tH J@J Heading 1$7$8$@&H$56CJ\]aJ<A@< Default Paragraph Font6<KS>?H S}M\3EY@>3s| + m   "   Z G a T y #Ad-o a6e$2q34Iqr#Ex5800000S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S 0S 0S 0S 0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S0S 0S 0S 0S 0S 0S 0S000"` 6""ch#3} yIXgwEKeubhhn e n | : C   .7uy qz'08=Hu{@B>Htym o @ G N T V #( djosah68$&qzILr~83333333333333333333333333333333333333333333333Holly Dressman@C:\Documents and Settings\dress002.GENETICS\My Documents\hyb.docHolly Dressman@C:\Documents and Settings\dress002.GENETICS\My Documents\hyb.docHolly Dressman@C:\Documents and Settings\dress002.GENETICS\My Documents\hyb.docHolly Dressman@C:\Documents and Settings\dress002.GENETICS\My Documents\hyb.docHolly Dressman@C:\Documents and Settings\dress002.GENETICS\My Documents\hyb.docHolly DressmanDC:\Documents and Settings\dress002.GENETICS\My Documents\SOP hyb.docHolly DressmanDC:\Documents and Settings\dress002.GENETICS\My Documents\SOP hyb.docHolly DressmanDC:\Documents and Settings\dress002.GENETICS\My Documents\SOP hyb.docHolly DressmanWC:\Documents and Settings\dress002.GENETICS\Desktop\For CD TRCCRP standards\SOP hyb.docHolly DressmanWC:\Documents and Settings\dress002.GENETICS\Desktop\For CD TRCCRP standards\SOP hyb.docQzU%_+2tt^`o(.^`.pLp^p`L.@ @ ^@ `.^`.L^`L.^`.^`.PLP^P`L.^`o(.^`.pLp^p`L.@ @ ^@ `.^`.L^`L.^`.^`.PLP^P`L.QzU%_+2                  8@| | | |   VVVV6`@``@``$@```0@``8@``@@UnknownGz Times New Roman5Symbol3& z Arial"qhvh&h&f&[# (!202QStandard Operating ProcedureHolly DressmanHolly DressmanOh+'0 0< X d p |Standard Operating ProcedureMitanHolly Dressmaniolloll NORMAL.DOTmHolly Dressmani3llMicrosoft Word 9.0P@F#@bO@LuM@M[#՜.+,0 hp  Duke Univ. 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