The following procedure was developed by Hongbing Tang in our lab. Lysolecithin permeabilizes the plasma membrane. A short treatment of salivary glands with this reagent renders the glands permeable to DNase I. This allows one to probe for protein DNA interactions with DNase I.
The following stock solutions are made ahead of
time:
| Dissection
buffer
150 mM Sucrose 80 mM KCl 35 mM HEPES pH 7.6 5 mM MgCl2 0.5 mM CaCl2 |
Digestion
buffer
150 mM Sucrose 30 mM Tris-Cl pH 7.6 60 mM KCl 15 mM NaCl 5 mM MgCl2 2 mM CaCl2 |
| Lysis buffer
20 mM Tris-Cl pH 7.6 20 mM NaCl 40 mM Na2EDTA pH 8.0 1% SDS |
Dissolve 5 mg of lysolecithin in 5 ml of Dissection buffer to make lysolecithin buffer. Store this solution at 4oC.
A freshly made solution is clear but becomes cloudy upon storage at 4oC. The cloudy solution works well but must be vigorously shaken before use. The lysolecithin remains effective for at least several weeks if stored at 4oC.Prepare 200 ul aliquots of 0, 50 U/ml, 100 U/ml, 200 U/ml, and 400 U/ml DNase I solutions by combining DNase I stock (10,000 U/ml or higher) with Digestion buffer. Mix by triteration and store on ice.
For each digestion condition, dissect salivary glands from 6 larvae and place the glands in a 1.5 ml siliconized microfuge tube containing 100 ul of ice cold dissection buffer. Perform dissections in the dissection buffer listed above. Complete the dissection, digestion and lysis of one digestion condition before starting another.
After collecting all the glands together in 100 ul of cold dissection buffer, add 100 ul of cold lysolecithin buffer. This gives a final volume of 200 ul with a concentration of lysolecithin at 0.5 mg/ml. Incubate the sample for 4' on ice.
Remove the buffer from the glands and add 200 ul of a DNase I solution. Incubate at 25oC for 3'.
Add 200 ul of lysis buffer and follow with an addition of 20 ug of proteinase K.
Incubate the sample at 37oC for 60'.
Extract the sample with a sequence of equilibrated phenol (pH 8), equilibrated phenol (pH 8), and chloroform. Each extraction is done with 400 ul of the organic solution. Samples are shaken for 5' and centrifuged for 5' to separate phases. For both of the phenol extractions, the aqueous phase is transferred to a fresh unsiliconized tube. For the chloroform extraction, the chloroform layer is removed by pipetting it out from underneath the aqueous phase.
Add 40 ul of 3M Na acetate pH 7 and 1 ml of cold Ethanol. Thoroughly mix by inverting the tube and place on ice for at least 15'. Microfuge in the cold for at least 20'. A small pellet should be evident. Discard the supernatant and add 100 ul of cold 75% Ethanol. Microfuge in the cold for 5' and discard the supernatant. Allow the samples to air-dry.
Dissolve the nucleic acid pellet from the salivary glands in 20 ul of TE (10 mM Tris pH 7.5, 1 mM EDTA).
Determine the concentration of DNA by measuring 1 ul of the sample on the Fluorometer.
Store samples in the refrigerator for short term or in the freezer for long term. Analyze 100 ng by LM-PCR. For a description of the LM-PCR procedure, consult the permanganate footprinting of salivary glands.