By Albert F. Blakeslee
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Extra resources for An Apparent Case of Non-Mendelian Inheritance in Datura Due to a Disease
A less expensive solution is to grow and stain cells directly on 35 or 60 mm tissue culture dishes that can then be observed from the top with upright microscopes after mounting samples with glass coverslips. Microscopy with high numerical aperture immersion objectives requires a short working distance to the object ($200 mm); hence, high resolution microscopy is not possible through the cell culture dish bottom or through microscope slides ($1,000 mm). 1. Wipe 22 Â 22 mm glass cover slips; clean using 100% acetone.
6. Incubate with diluted secondary antibodies at room temperature for 60 min. 7. 02% TX-100 at room temperature for 15 min, Â 3. 8. Rinse once with distilled water. 9. Mount samples with one drop of PVA or any other suitable mounting medium (see Note 12). Polymerize PVA at room temperature overnight. Store samples at 4°C afterwards for several months or years. 5. Immunostaining of Living Cells for Fixative-Sensitive Extracellular Epitopes It is often advantageous to incubate living cells with primary antibodies prior to the fixation, if antigens are sensitive to fixatives or organic solvents or when using antibodies that only recognize native protein configurations.
CDNA was synthesized using a random hexamer, and amplicons were separated on a 1% ready agarose gel (Bio-Rad). Positive as well as negative controls had been included in the amplification experiments. FFPE: formalin-fixed and paraffin-embedded tissue; NCT: negative control (without cDNA); PCT: positive control (fresh rat kidney); M: ready-loadTM 100 bp DNA ladder (Invitrogen). 2. Carry out PCR amplification for 35 cycles in the PCR machine 9700. 3. To confirm the size of PCR amplicons, carry out agarose electrophoresis using 1% ReadyAgaroseTM Mini Gel with 1 Â TAE buffer.
An Apparent Case of Non-Mendelian Inheritance in Datura Due to a Disease by Albert F. Blakeslee