Hybridization ovens

The temperature of the hybridization oven is reduced to 25-30°C. The wash solution is replaced by low-salt wash solution 2 to approx. 80% capacity of the container and rotated for 5 min. 

For the hybridization step, we employed a Dako hybridizer however, we also tested the protocol using a conventional hybridization oven and obtained similar ISH results. We applied 50 pi hybridization mix and sealed with a cover glass. In this experiment we did not seal the cover glass with rubber cement, which is proposed when incubating smaller hybridization reagent volumes (38, 40). 

Change Hybridization Mix and incubate the embryos for 1 h at 70°C in the hybridization oven with rocking. 

Prepare WASHl solution in the 12-well plate, and transfer the embryos with mesh-buckets. Wash 3 times with prewarmed WASHl solution at 70°C in the hybridization oven for 30 min each see Note 15). Probes can be recovered in the screw-cap tubes and stored at -20°C for reuse up to at least 3 times. 

Wash twice with prewarmed WASH2 solution at 65°C in the hybridization oven for 30 min each. 

The heating phase for melting the powder is crucial to the final surface quality. The heat-up should be fast but uniform. The ovens used to melt the powder on the part can be convection, infrared, or combination (hybrid) ovens. Infrared (IR) or combination (IR/convection) ovens are most widely... 

Hybridization oven containers or blot bags and heat sealer. 

Hybridization oven, heated water bath, or air shaker. 

Add 50 ng 32P-labeled DNA probe to carrier DNA and heat to 100°C for 10 min. Add to 10 mL hybridization mix (3X SSC, 2% SDS, 5X Denhardf s, 0.2 mg/mL sheared carrier DNA). Remove prehybridization solution and replace with hybridization solution containing probe. Incubate in hybridization oven or shaker overnight at 65°C. 

Preheat wash buffer (0.1% SDS/0.1X SSC) to 65°C. Remove filter from bag and place in plastic box. If using hybridization tubes, carry out initial washes in tube and then transfer to plastic box. Rinse twice with approx 500 mL wash buffer. Wash blot three times for 30 min in 1 L of wash buffer. Wash in shaker, water bath or hybridization oven at 65°C. 

Special hybridization ovens or convenient acrylic cassettes to be placed in a waterbath are commercially available. The membrane is placed in the hybridization cassette (e.g., from Bios Corp., New Haven, CT 06511 Fax (203) 773-0017), covered with (pre)hybridiza-tion solution, the top closed over a cover and incubated at the desired temperature. Hybriboxes can also be constructed as reported by Cohen et at. (1990 Section 7.2.6). The hybridization... 

To each bottle containing an array membrane, add 3-5 ml of pre-warmed Hybridization Buffer (provided). Place each bottle into a rotating hybridization oven, preheated to 42 °C, for 2 h. 

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. 

The incubation of the elution buffer with the Dynabeads can be done on a heat block (as written in the Panomics protocol) or in a water bath as well. The use of the hybridization oven is also convenient if it is not being used to hybridize the membranes at the time. 

Remove GeneChips from 4°C storage. Allow chips to warm to room temperature prior to hybridization (allow at least 60 min for GeneChips to equilibrate). Likewise, thaw reagents from cold storage at room temperature. Set heat block at 65 °C and set the hybridization oven to 45 °C. 

Remove the GeneChips from the hybridization oven and place a 200-pL pipette tip into one of the septa. Remove the hybridization cocktail and place into the appropriate sample tube. These samples can be run on other chips at a later date. Store unused samples at -80 °C. 

Hybridization oven with rotor and cylindric glass bottles (30 x 4 cm) nylon meshes (Hybaid, Woodbridge, NJ) and freezer boxes, large enough for 20 X 20 cm blots see Note 3). 

If hybridization is performed in a box, then the blot must be placed on top of the buffer and allowed to saturate fully before being submerged. This prevents the appearance of white patches on detection. A volume of buffer equivalent to 0.25 mL/cm of membrane should be used. If the box is significantly larger than the blot then the volume used should correspond to the area of the bottom of the box. Hybridizations may also be carried out in bags or in hybridization ovens. In these cases the blot should be put into the container first. Buffer is added at a volume equivalent to 0.125 vaUcvc of membrane. 

Microarray platform For protocols used here, computer workstation Type I with GeneChip operating software (a Windows XP-based unit with pre-instaUed specialized controller boards to control the GeneChip Scanner 3000 and Fluidics Station 450) (Xl-0180, hybridization oven 640 800139, GeneChip Huidics Station 450 00-0079, GeneChip Scanner 3000 7G Systan (X)-0213. 

Immediately place the tnbe in a 37°C water bath, hybridization oven, or thermocycler in which the heated lid parallels the block temperature. Incubate for 16h. Periodically mix the components by gentle pipeting and pulse to collect condensation. We generally leave this reaction to run overnight in a 37°C water bath with a sealed lid. 

Equilibrate the GeneChips (stored at 4°C) to room temperature. This is essential to prevent subsequent rupturing of seals and septa (approximately 20min). Set the hybridixation oven (Affymetrix GeneChip hybridization oven 640, 800138). 

During this incubation, remove the liquid from inside chamber of GeneChip (approximately 200pL) and replace with 200pL of lx hybridization mix. Incubate in the hybridization oven at 45°C, 60rpm for lOmin (see Notes 37 and 38). The remaining mix can be stored at -20 C. 

Change solution for hybridization buffer prewarmed to 60 °C incubate at 60 °C for at least 1 h, with horizontal rotation. A hybridization oven is useful. 

FIGURE 1 Incubation of a Northwestern blot in an hybridization oven. 

Pour out the buffer from the hybridization bottle and replace it with 5 ml of the labeling probe. Place in a hybridization oven and leave it to rotate for 1 hr at room temperature. If plastic bags are used, tape them onto a rotating wheel. 

Hybaid Maxi 14 hybridization oven (Thermo Electron, UK). 

It is occasionally found that modifications can be made to conventional processes by introducing an intensification method that operates in parallel (or in series) with the conventional process. The research carried out by EA Technology in the UK resulted in a hybrid oven for firing ceramics, bringing together conventional gas firing and intensified microwave heating of the ceramic ware (Anon, 2000). 

Ceramics manufacture Electric fields - microwave hybrid oven... 

When the cultures in the 24-well plates have reached confluence, aspirate the medium and wash once with PBS Add 0 5 mL of digestion buffer and incubate in a humidified atmosphere at 60-65 C overnight Humidity is best generated by placing the tissue-culture plates in an airtight container (e.g, Tupperware) containing saturated paper towels The container is then placed in a suitable oven (hybridization ovens work very well for this purpose). 

Hybridisierungsinkubator hybridization oven Hybridisierungsofen hydrate Hydrat hydration (solvation) Hydratation, Hydratisierung, Solvation (Wassereinlagerung, W asseranlagerung) hydration shell Hydrathtllle,... 

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