By Andrzej Boguslawski
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Extra resources for A Note on Two Dogmas in Pragmatics
Mark the posrtron of the sample wells on the mtrocellulose and label each filter strip. Soak the filter m 2 x SSC for 10 mm, then bake them at 80°C for at least 2 h (see Note 7). Filters may be stored at 4°C for several months before hybridization Hybridization The filters are now incubated with a 32P-labeled sequence-specific probe. The probe is usually labeled by nick translation (see Chapter 38), and will associate with its complementary sequence on the filter. Filters are coated with Denhardt’s solution (4) and heterologous DNA before hybridization, to prevent nonspecrfrc bmdmg of the probe.
Dissolve overnight at 4”C, with gentle mrxmg 8. Add 100 PL of 20 X SSC and 10 PL of rrbonuclease, and incubate for 1 h at 37°C 9. Add 2 mL of sterile distilled H20, and extract the solution twice with chloroform-rsoamyl alcohol 10. Precrprtate the DNA by adding 2 vol. of absolute ethanol, and centrifuge at 5000g for 5 mm at 5°C. 5 mL of sterile drstrlled H20. 11. Scan a dilution of the DNA from 220 to 300 nm (see Note 3). Lg. Notes 1 DNA can be isolated from cultured cells as follows suspend cell pellet m tissue culture medium at a concentration of lo7 cells/ml Add 5 vol of cell lysrs buffer Mathew 34 and homogenize (Step 2).
Owing to the high density of loading buffer, care 1s needed to ensure that it mixes completely with the sample. 5M EDTA is included, this solution can double as a ‘stopping mix’ to arrest restnction endonuclease digestions. Samples are loaded mto the wells using a microplpet or mlcrosyrmge. With the syringe or plpet tip a couple of millimeters above the well, gently dispense the sample, which ~111 fall into the well. This method needs a reasonably steady pair of hands, but avoids any danger of accidentally mlecting the sample mto the gel beneath a well.