Standard saline citrate

Dip grids in 4X standard saline citrate (SSC) 0.15 MNaCl2, 0.015 M Na-citrate, sodium phosphate buffer, 7.0. 

Standard saline citrate (SSC), 20X concentration- 263 mM trisodium citrate, 3 08MNaCl. 

Standard saline citrate (IX SSC) 150 mMNaCl, 15 mM sodium citrate... 

Digoxigenin-labeled chromosome 10 a-satellite probe (DIOZI Oncor, Gaithersburg, MD) is used at a final concentration of 5 pl/100 pi in hybridization buffer (50% formamide, 10% dextran sulfate, 0.004% Tween 20, and standard saline citrate (SSC) [in 1.5 strength]). A 10-pl volume of the probe is placed on an 18 x 18-mm coverslip, which is placed onto the slide, sealed with special Vulcanizing Fluid, and placed on a flat-bed thermal cycler. Slides are incubated for 3 min at 94°C and placed in a humidified box for 16 hr at 37°C. 

Following shearing, dialyze the DNA at approximately 5° against 0.001X SSC (standard saline citrate 0.15 Af NaCl, 15 mAf sodium citrate, pH 7.0) and 1 mAf EDTA.11 The preparation can then be taken to dryness in a freeze-dryer, redissolved at 1 -5 fig/fi in distilled water, and frozen at —20° until needed. 

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)... 

RNAs are denatured by formaldehyde/heat treatment. All of the stock solutions for RNA slot blots are made using sterile diethyl pyrocarbonate (DEPC)-treated water. RNAs are diluted as necessary from stock solutions with water, and 3 volumes of 6.15 Mformaldehyde in 10X standard saline citrate (SSC) is added to give a final RNA concentration of 10-100pg/ml. The RNA dilutions are heated to 65° for 15 min and quick-chilled on ice. The denatured stock is further diluted with 4.16 M formaldehyde in 7.5 X SSC such that the desired concentration of RNA may be applied to each slot in a total volume of 400 pi. The nylon membrane is piewet in water and then soaked in 10X SSC for 20 min. Slot blots are performed using a commercially available apparatus hooked to a vacuum source. After the samples are blotted through, each well is washed with 400 pi of 10 X SSC. The membrane is removed from the apparatus and baked in a vacuum oven at 80° for 2 hr. 

X SSC (fivefold concentrated standard saline citrate) 75 mMNaCl, 75 mM trisodium citrate. 

Figure 3.1. A. A histogram of the DNA components of the bovine genome. The height of each bar is proportional to the percentage of each component in DNA solid bars correspond to the sharp-melting, open bars to the broad-melting components (see B). The broken line is an enlarged band profile of calf DNA in CsCl density gradient. B. Absorbance-temperature profiles for calf thymus DNA components in 0.1 xSSC (standard saline citrate). Compare the very sharp transitions of 1.705, 1,714 and 1.723 g/cm satellites with the broader ones of 1.697, 1.704 and 1.709 g/cm major DNA components. (From Filipski et al., 1973).

Slides are placed in hybridization trays containing Whatman filter paper soaked in standard saline citrate (SSC)/formamide solution. Riboprobe hybridization buffer is applied to tissue using a repeater pipet, and slides are cover slipped. The cocktail of digoxigenin and p S]-labeled ripobrobe hybridization buffer (or the [ Sj-labeled rihoprohe hybridization buffer alone) is appUed to tissue in fixed voiumes determined by the size of the cover slip required to cover the sections (i.e., 50 pL per 30-mm cover sUp for sections mounted one or two per slide or 100 oL per 5- mm cover sUp for sections mounted three or four per sUde). Slides are placed in a humidihed chamber and incubated overnight at 52-55°C. 

This will give T in 1 X standard saline citrate (1 x SSC), whereas these hybridizations are carried out in 0.28M PB, but the difference in T is not significant. 

Fig. 3. Thermal denaturation of poly I-poly C and the poly-L-lysine complex of poly I poly C (poly [iCLC]). The compounds, at a concentration of 50 yg poly I poly C/ml in 0.1 x standard saline-citrate, were heated to the indicated temperatures in a recording spectrophotometer set at 260 mm (T ).

FIGURE 5.6. Relation between sedimentation constant s 2o,w and molecular weight of DNA of different size. (After Refs. 5 and 6.) HMP, 0.01 M phosphate buffer SSC, standard saline citrate (0.15 M NaCI+0.015 M Na-citrate). 

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