By Mary A. Schuler and Raymond E. Zielinski (Auth.)
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Additional info for Methods in Plant Molecular Biology
You should observe two green bands. The upper contains stripped thylakoids (the remains of chloroplasts with broken envelope membranes) and the lower intact chloroplasts. Remove the bands from the gradient using a Pasteur pipet and collect them separately in fresh centrifuge tubes on ice. Be sure to mark the centrifuge tubes to indicate which band is which! Dilute the chloroplast suspensions with about 30 ml of GR medium. Cap the tubes tightly with Parafilm and mix by inverting the tubes several times.
Dry filters on a paper towel at room temperature. 18. Scotch tape filters onto a small sheet of filter paper. Mark the edges of the filters asymmetrically with 32P-labeled India ink (ink plus a little [32P]dATP). 19. Put Saran wrap over filters so they don't stick to the film. Put them on XAR-5 film and expose in -70°C freezer for 1 day. EXPERIMENT 2 20. Take filters off film and develop X-ray. At this point you should have at least a few colonies that have hybridized with your probe. " 21. Inoculate small YT cultures as outlined at beginning of the "miniprep" method and grow cultures overnight at 37°C.
Place filters in a 75°C oven for 1 hour in order to bake the DNA onto the filters. 2% sarkosyl, l x Denhardt's solution, 50% formamide) per filter using a 10-ml syringe to add the solution through a corner of the bag; seal bags; check for leaks; submerse bags in a 42°C water bath for at least 8 hours. Now we will skip to the nick-translation procedure since this can be done while your filters are baking. 26-kb HmdIII fragments cloned in your pUC vector. The nick-translation procedure should be done behind an isotope shield.
Methods in Plant Molecular Biology by Mary A. Schuler and Raymond E. Zielinski (Auth.)