Hybridization washes

Following hybridization, wash the membrane 2x15 min in Hybridization Wash 1 and 2x 15 min in Hybridization Wash 2 at 57°. 

Following hybridization, wash slide with gentle agitation in 50% formamide 2X SSC at 37°C for 12 min. The cover slip should dislodge. 

A typical transcript analysis starts with isolation of total RNA, followed by cDNA synthesis and labeling. Next, the pnrifled and labeled cDNA is applied onto a microarray that contains thon-sands of immobilized probes, hybridized, washed and scanned, and the signal for every probe estimated and analyzed (Fig. 2). 

Cellular DNA is extracted, resuspended, restriction enzyme digested, and elec-trophoresed in a 1% agarose gel, and transferred via Southern blot technique to nitrocellulose membranes. The nitrocellulose is baked, prehybridized, hybridized, washed, quantitatively analyzed for radioactivity, and exposed to film. 

To remove excess probe, heat hybridization wash buffer (0.1X SSC/ 0.1% SDS, 600-750 mL/filter total) to 60-65°C. Remove the membranes from the hybridization mix, rinse each one with 5-10 mL of wash buffer from a squirt bottle, and place them immediately in a container of wash buffer (200-250 mL/filter). Two or three membranes may be washed together. Do not let membranes dry before washing is complete. 

The LiPA control should show reaction on lines 1, 2, 3, 6, 11, 13, and 16. The LiPA control consists of biotinylated oligonucleotides complementary to the hybridization probes applied on these lines. Positive hybridization to these lines demonstrates satisfactory performance of the assay including hybridization, washing, and detection. The amplification control sample gives a clear positive signal on gel and on the strip on lines 1, 2, 3, 6,11,13, and 16. 

After hybridization, wash the membranes ten times with 1 x SSC, containing 0.5% SDS, at 50°C and five times with 1 X SSC. Wash thp membrane once with 5 mM Tris-HCI (pH 7.5) and 1 mM EDTA (30 s) and elute by boiling for 5 min with 5 mM KCl, 2 mM EDTA and 10 p-g/ml tRNA and collect the supernatant. Repeat and pool the eluates. 

During the FISH procedure, DNA within cells placed on a slide and the fluorescently labeled probe of interest are denatured by incubation at high temperature. Then the probe is allowed to hybridize to the target DNA. The next step is a series of post-hybridization washes to remove the probe excess. Finally, after counterstaining the nuclei, the probe signal can be visualized under a fluorescent microscope. 

Hybridization Wash Buffer I distilled H2O (RNase, DNase free), 20% SDS, 20x SSC. These reagents are found in the Panomics TranSignaF TF-TF Interaction Array Membranes and Hybridization Reagents (Box 1). 

Before finishing the overnight hybridization, prepare Hybridization Wash I and Hybridization Wash II for use in step 6 (Hybridization Wash Buffer I 262.5 ml Distilled Water, 7.5 ml 20% SDS, 30 ml 20x SSC. Hybridization Wash Buffer II 291 ml Distilled Water, 7.5 ml 20% SDS, 1.5 ml 20x SSC). Pre-warm each Hybridization Buffer in a water bath at 35-50 °C. 

To wash each membrane, add 50 ml of pre-warmed Hybridization Wash I and incubate at 48 °C for 20 min in the rotating hybridization oven. Decant the liquid and repeat the wash. Decant the second wash and add 50 ml of pre-warmed Hybridization Wash n. Again, incubate at 48 °C for 20 min in the rotating hybridization oven. Decant the liquid and repeat the wash. 

After hybridization, wash the sections for 20 min in 3X SSC at room temperature (during this first wash, coverslips will float away). 

Precautions should be taken to ensure that all solutions, spatulas, plasticware, and glassware are sterile and free of any dust throughout the procedure. All materials must be RNase-free for all stages between the initial fixation and the post-hybridization washes (see Note 1). 

Goat or sheep serum thaw, aliquot, and store at -20°C until day of post-hybridization washes. 

In contrast to all steps prior to hybridization, washes can be performed with nonsterile material, except for the O.IX SSC final steps, because RNA-RNA hybrids are sensitive to RNases at low-ionic strength... 

Prehybridization solution 5X SSPE, 5X Denhardts, 1 mg/mL ssDNA, 0 1% SDS Hybridization solution 5X SSPE, IX Denhardt s, 1 mg/mL ssDNA Hybridization wash solution 2X SSPE/0 1% SDS GeneClean II kit (Bio 101, Vista, CA). 

Following hybridization, wash the blots twice for 10 mm each in 2X SSC, 0 1% SDS at room temperature followed by washing at 60°C as required (see Subheading 3.6., step 7) in 0 IX SSC, 0 1% SDS Expose blots to Kodak X-Omat AR autoradiography film overnight at -70°C... 

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