Prehybridization solutions

For prehybridization, place pairs of filters containing lysed, fixed colonies back to back in sealable plastic bags. Add 20 mL of prehybridization solution, seal the bag, seal it within a second bag, and incubate at 42°C for 2 h. Maintain the proper temperature by submersion in a water bath. 

After fixing DNA onto the filters, prehybridize them in a smooth-bottom photo tray (25 X 25 cm). For eight filters, prehybridize in about 600 ml of prehybridization solution and incubate at 65°-68°, with shaking (50 rpm), for at least 2 hr. Cover the photo tray with plastic wrap and a glass plate to prevent evaporation. 

Hybridize the eight filters in about 200 ml of hybridization solution that contains the denatured 32P-labeled probe [specific activity, 1 X 10 counts/min (cpm)//ig] of at least 10s cpm/ml. Denature the probe by adding volume of 5 M NaOH, then incubate at 68° for 15 min followed by the addition of volume of 5 M HC1 and volume of 1 M Tris-HCl (pH 7.4). Mix, then quickly add filters to hybridization solution that contains 50 /ig/ml poly(A) (Sigma, St. Louis, MO, P-9403), which is used to reduce nonspecific binding of the probe to the filter. Pour off the prehybridization solution and add the hybridization solution incubate at 68° with shaking for at least 12 hr or overnight. 

Hybridization solution Prehybridization solution plus 50 /ig/ml poly (A) (Sigma, P-9403), note that 20 /ig/ml denatured sonicated calf thymus or salmon sperm DNA can be used in place of poly(A). 

Blots are prehybridized to block all nonspecific binding (which causes background). To do this, one first wets the nitrocellulose in 3 X SSC, then in 50% formamide, 3X SSC, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA (pH 8.0), 10 mM Tris (pH 7.5), 10X Denhardt s solution [1X Denhardt s is 0.02% Ficoll, 0.02% bovine serum albumin (BSA), and 0.02% polyvinylpyrrolidone 360], 0.05% sodium pyrophosphate, and 100 /ig/ml denatured DNA. Denatured DNA is sheared herring or salmon sperm DNA that has been boiled for 10 min before addition to the prehybridization mix. Blots are sealed within plastic bags, placed between two glass plates to give uniform distribution of prehybridization solution over the blot, and incubated in a water bath at 37° for at least 1 hr. 

Prehybridization solution 50% formamide, 5X SSC, 5X Denhardt s solution, 25 mMsodium phosphate, pH 6.5,300 pg/mL freshly denatured sheared herring sperm DNA. Filter through Whatman no. 1 paper on a Buchner funnel, then through a sterile 0.45-pm filter. Store 10-mL aliquots in glass at -20°C. Use only once. 

Hybridization solution 45% Formamide, 5X SSC, 5X Denhardt s solution, 20 mMsodium phosphate, pH 6.5, 300 pg/mL freshly denatured, sheared, herring sperm DNA, 200 ng of biotinylated DNA/mL. Before its addition, the biotinylated probe DNA is denatured by incubating for 10 min in a boiling water bath and quickreduce size is unnecessary, since the products generated by nick translation are sufficiently small. Filter and store the hybridization solution as was done for the prehybridization solution. Hybridization solution can be recovered after use and stored at-20°C. The solution can be reused at least 10 times over a time-span of at least 5 mo, without noticeable... 

For prehybridization, place pairs of filters containing lysed, fixed colonies back to back in sealable plastic bags. Add 20 mL of prehybridization solution, seal the bag, seal it within a second bag, and incubate at 42°C for 2 h. Mainbun the proper temperature by submersion in a water bath. After prehybridization, replace the liquid with 20 mL of hybridization solution, exclude air bubbles, reseal the bags, and immerse in the water bath. Brief incubations (1 h) are suflBcient for the detection of relatively abundant sequences, such as unamplified plasmid pBR322 in K cok More extensive incubations (45 h) may be necessary to detect less abundant sequences. 

Prehybridize the Southern membranes for a few hours or overnight in a sealed polythene box containing sufficient prehybridization solution to allow free... 

Replace the prehybridization solution with hybridization solution containing approximately 10 counts min" ml of purified, labelled DNA probe. Allow hybridization to proceed at 55°C (for M13) or 62°C (Jeffreys probes) overnight with shaking. 

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. 

Before prehybridization, nitrocellulose should be wetted with 1 X SSPE (or 1 X SSC), containing 0.1% SDS, whereas nylon membranes can be used directly. Prehybridization and hybridization is performed in polythene bags, plastic boxes (hybridization cassette) or hybridization incubators (Section 8.1.2). The cassettes and incubators are economical in the use of hybridization solutions. For plastic bags, use 0.25 ml/cm for nylon membranes and 0.1 ml/cm for nitrocellulose membranes of prefiltered (important ) prehybridization solutions. The bags should be massaged to completely distribute the solution and incubated with agitation. 

Prehybridization solution is removed from the bag and the hybridization solution is added but probe may be added directly to prehybridization solution (Section 8.1.2). Hybridization buffers should match those used for prehybridization in the case of radiolabeled probes and nitrocellulose membranes. The volume of the hybridization solution should be kept as small as possible. The probe is evenly distributed by massaging the bag and incubating at the proper temperature with agitation. The probe should be clean (spin column, Cameo IIS or Unlflo cellulose acetate filters for radioprobes) and denatured, if necessary, by boiling in TE for 5 min (radioprobes. 

Prefilter the sample through preblocked (two 0.2 ml aliquots of prehybridization solution) Teflon acrodiscs (Gelman, 0.2 xm) and filter slowly (50 j,l/min) through a preblocked poly(dT)4ooo nylon membrane (prepared by immobilizing poly(dT), filtered trough an acrodisc, as described in Table 8.5). Determine (radio)activity on membranes. 

Fix by baking and detect DNA (Section 8.2). Scrape bacterial debris from the membrane in prehybridization solution (with a glove). 

Remove prehybridization solution without permitting the specimens to dry and add probe in hybridization solution (radiolabeled probes 10—600 ng/ml nonradioactive probes up to 10 times more). Hybridization solution contains 50% formamide, 0.5 M NaCl, 10 mM Tris-HCI (pH 7.6), 2 mM EDTA, 1 x Denhardt s solution, 20 mM DTT, 300 pg/ml tRNA, 300 (xg/ml poly(A) or/and poly(C) (optional), 10% PEG 6000 RNase-free. S label may form nonspecific disulfide bonds in the cell if reductants, such as DTT or 2-mercaptoethanol, are omitted. 

If the sample is on a slide Pipet the probe(s) in 11-15 p hybridization solution onto a clean 18 x 18-mm or 22 x 22-mm cover slip (depending on how much area the specimen covers). Remove a slide from the prehybridization solution (wear gloves, because formamide is toxic). Remove as much of the liquid as possible by wiping the edges and aspirating carefully around the sample. Touch the specimen to the drop of hybridization solution to pick up the cover slip. Quickly invert the slide the probe solution should spread out under the cover slip. 

If the sample is on a cover slip, do the reverse Pipet the probe solution onto a clean microscope slide. Remove the cover slip from the prehybridization solution and blot the back side carefully on Kimwipes. Touch the drop of probe solution to the sample to pick up the cover slip and invert. 

Prehybridize the blots at 60-65°C in either plastic bags or plastic boxes for 30 min using 3 mL of prehybridization solution/100 cm. ... 

Replace the prehybridization solution with fresh hybridization solution containing the denatured probe. Incubate from 6 h to overnight at 60-65 C. Mixing during hybridization is not necessary. 

Quench the probe in iced water for 5 min see Note 4) and then add this to the unused prehybridizing solution at 42 C and mix thoroughly. 

If the film has a uniformly high background, then the blocking washes are likely to have been inadequate, or the membrane used is highly charged and requires the addition of more SDS (up to 0.5%) in the prehybridization solution. 

Add the labeled oligonucleotide probe to the prehybridization solution to give a final concentration of 5-10 ng/mL see Note 2). 

Remove the prehybridization solution and add 0.5-1.0 mL of hybridization solution plus tRNA and DNA containing a 1 100-1 200 dilution of freshly denatured digoxigenin-labeled RNA probe (1.0-1.5 ng/pL). If using the Pierce Reacti-vials, place a foam rubber insert containing an open 0.5-mL Eppendorf tube filled with hybridization buffer into the neck of the vial (but well above embryos) to humidify the chamber. 

Prehybridization solution consists of the following and should be prepared on the y of use 50% deionized fonnamide, 5xSSC, 2% blocking powder, 0.1% Tween-20,0.5% CHAPS, 50 J.g/mL yeast RNA, 5mM EDTA, 50 j,g/mL heparin. Bovine serum albumin (Fraction V Sigma-Aldrich A9467). 

Prehybridize embryos in 1 mL of prehybridisation solution at 65°C (3h). Embryos can be stored at this point at -20°C in prehybridization solution. 

Transfer the filters to a hybridization bag, and add 2 mL/filter of Church s buffer (7% SDS, 0.5 M NaPO, pH 7.2) (16). Prehybridize at 65°C for a convenient time, usually 15 min to several hours. Up to 20 filters (132 mm) can be processed/bag. Remove the prehybridization solution and add fresh Church s buffer containing 5% w/v Dextran sulfate (Pharmacia) at 750 pL/filter. Denature the probe by adding 0.1 vol 2 N NaOH and incubating at room temperature for 5 min. Add the denatured probe directly to the bag, seal, and mix well. Hybridize at 65°C overnight with shaking if possible. 

Immerse the slide to be hybridized in 50mL lx prehybridization solution. A 50-mL falcon tube is a convenient container for this procedure. 

Incubate the slide in this prehybridization solution at 42°C for 30min. 

Replace with fresh prehybridization solution and incubate at 65-70°C for 1 h-over-night. We place samples in Eppendorfs in a hot block where possible, to minimize probe volume (0.5 mL is sufficient). Large samples can be placed in multiwell dishes in an oven. At this point, samples can be stored at -20°C for a period of months. 

Incubate embryos with prewarmed hybridization solution containing approximately Ipg/mL of riboprobe (usually 10 pL probe to ImL prehybridization solution). For double in situs, add both DIG- and FlTC-labeled probes simultaneously. Incubate at 65-70°C overnight. 

Prehybridize the filter in prehybridization solution at 42°C in a shaking water bath for at least 3 h. 

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