Hybridization prehybridization

Some or all prehybridization steps are optional, depending on the target and specimen. Prehybridization entails soaking the slides containing the specimens in a prehybridizafion buffer in order to have the specimen rehydrate and equilibrate with the hybridization buffer, to be used later. The prehybridization phase may also entail treatment with proteases or nucleases in order to improve probe access and to reduce background. 

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 sufficient for the detection of relatively abundant sequences, such as unamplified plasmid pBR322 m E. coli. More extensive incubations (45 h) may be necessary to detect less abundant sequences. 

Southern Hybridization. Our hybridization procedures are essentially those of Palmer.3 New blots are washed for 1 hr at 65° in 0.1 X SSC, 0.5% (w/v) sodium dodecyl sulfate (SDS) blots that have been used previously are treated with 0.4 N NaOH at 40° for 30 min and rinsed 3 times with 2 X SSC for 10 min each time. If a large number of blots are to be hybridized simultaneously, 10 can be sealed into one bag and 50 ml of hybridization buffer added. Hybridization buffer is 4X SSC, 10 M EDTA, 5X Den-hardt s [50 X Denhardt s is 1.0% (w/v) BSA, 1.0% (w/v) Ficoll 40 (Sigma), and 1% (w/v) polyvinylpyrrolidone (PVP type 400, Sigma)], 0.5% (w/v) SDS, and 25 /tg/ml of calf thymus DNA sonicated and freshly boiled. All except the last component are Millipore (Bedford, MA) filtered before the DNA is added. For heterologous probes we prehybridize and hybridize at 55° for homologous or highly conserved DNA we use 65°. We prehybridize for a minimum of 2 hr, then replace the buffer with fresh solution. The denatured probe DNA (see below) is diluted in 5 ml buffer and added. Probe concentrations should not exceed 50 to 100 ng/ml for buffers that do... 

If the target sequence is present on the filter in a low concentration, either because it is a low copy number sequence or because a particular group of taxa yield a low concentration of chloroplast DNA in a total DNA extract, a prehybridization/hybridization buffer that contains dextran sulfate can greatly enhance the rate of hybridization (up to 100-fold44 when nick-translated probes are used) and permit detection of the target se-... 

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). 

Prehybridization/hybridization buffer 50% deionized formamide, 5 X SSC (standard saline citrate), 8 X Denhardt s solution, 50 mM sodium phosphate buffer (pH 6.5), 0.5% sodium dodecyl sulfate (SDS), 250 fig/ml denatured herring testes DNA, and 500 ng/ml yeast RNA (for the composition of SSC buffer and Denhardt s solution, see, e.g., Ausubel et al.13)... 

Hybridization Procedure. Formamide is deionized by adding 1 g of mixed bed ion-exchange resin (e.g., AG 501-X8, Bio Rad, Hercules, CA) to 10 ml of formamide, which is stirred on a magnetic stirrer for at least 1 hr and then filtered twice through filter paper. Store at —20°. Prehybridize filters in the above buffer for 4-8 hr at 45°, replace the solution with fresh... 

After Southern transfer of restriction-digested genomic DNA, filters are prehybridized for 1-4 hr. The probe is boiled for 10 min to denature it, added to fresh hybridization buffer, and hybridized overnight. To calculate appropriate hybridization temperatures in buffers with or without forma-mide, and for details of hybridization buffers and washes, see standard protocols.13 For hybridizations at low stringency to detect signals from fragments of approximately 65-70% overall sequence similarity, we apply 55° for hybridization and washes in 6X SSC without formamide. Similar protocols can be used for colony and plaque hybridizations. 

Blots are hybridized under the same conditions as for prehybridization with the addition of denatured labeled probe. The labeled probe has a specific activity of at least 0.5 X 108 counts/min (cpm)//ig and is present at a concentration of at least 1 X 106 cpm/ml. The probe is denatured by treatment with 1/100 volume of 10 ANaOH for 10 min at 68° followed by neutralization with 1/10 volume of untitrated 1.5 M NaH2P04. The blots are hybridized for 40-48 hr at 37° between glass plates in a water bath. 

The reagents and buffers required for in situ hybridization techniques are diverse, particularly for a method using immunocytochemical detection. The following sections have therefore been subdivided into prehybridization, hybridization, and posthybridization. Genomic human DNA was extracted from fetal placenta according to the method described in Maniatis (13) and the alphoid X-centromere specific probe was as described by Wolfe etdl (14). 

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... 

M13 multilocus method. Prehybridization and hybridization solution 0.263 M disodium hydrogen phosphate, ImM EDTA (pH 8.0), 7% sodium dodecyl-sulphate (SDS) and 1% bovine serum albumin (Fraction V, Sigma)... 

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. 

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. 

Incubate nitrocellulose membranes briefly (15 min) at 42°C in prehybridization buffer (incubations overnight will not harm the membranes). The authors normally prehybridize for only the amount of time required for the denaturation of the labeled hybridization probe (see step 4). 

Remove the prehybridization buffer from the membranes, add the hybridization buffer (with labeled fragment), and incubate at42°C for 36-72 h. (Shorter times, such as 18 h, are acceptable for slots or rehybridization however, 40-45 h is optimal for initial hybridizations). 

Hybridize for 16-20 h at 65°C in fresh prehybridization buffer containing EBV BamHI-W fragment labeled with 32P by random primer kit (Stratagene). 

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. 

Five steps can be distinguished in membrane hybridization (i) immobilization of target nucleic acid (ii) prehybridization to saturate the remaining binding sites which would otherwise adsorb probe non-specifically (iii) hybridization in low stringent conditions to adsorb probe as efficiently as possible (iv) posthybridization washes to define the stringency of the hybridization and thus the specificity of the reaction (v) the detection step (Fig. 8.2). In addition, the hybridized probes can sometimes be stripped from the blots to expose the targets to other probes. 

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. 

As a general rule, prehybridization conditions (buffer and temperature) are, except for ohgonucleotide probes and charged nylon, identical to those of hybridization although the time is shorter (usually fast but prolonged prehybridization does not harm). Most (pre)hybridization buffers are variants of two basic buffers, one with formamide and one without. Formamide allows the use of lower incubation temperatures and an easy adjustment of the stringency and is generally used, or even essential, for RNA hybridization or for nonradioactive probes. It is recommended, particularly when formamide is used, to include a buffer, such as sodium/potassium phosphate, Tris-HCl, or PIPES-NaOH, or to use SSPE instead of... 

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. 

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