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Additional resources for Arthritis Research: Volume 2: Methods and Protocols (Methods in Molecular Medicine)
In order to make sure that the cell is transferred into the tube, the tip of capillary 2 may be broken into the tube and examined under the microscope afterwards. 11. PCR tubes are centrifuged shortly and are stored at –20°C. 12. Sections can be used for micromanipulation for up to 12 h without dramatic decrease of PCR efficiency (see Note 6). 4. 1. Preparation of PCR Templates 1. To prepare DNA for PCR, add 1 µL of Proteinase K solution (16 mg/mL) to each of the tubes containing isolated cells.
Reduce background. Even after enrichment your sample likely contains residual cells generating a background by carrying surface bound λ-chains. , step 1) in a quantitative manner. Run controls. , omitting the incubation with the catch-antibody should inhibit the staining completely). , step 2 and go on with two vials, with and without the catching antibody. Perform counterstainings for characterizing your antibodysecreting cell population for contaminants. Titrate reagents. In order to obtain optimal spots clearly separated from each other, titrate the reagents and vary incubation times, if necessary.
10. PCR efficiency is variable depending on the tissue condition and on the efficiency of cell isolation. In our hands, the efficiency of single-cell amplification (defined as percentage of cells for which at least one PCR product was obtained) was in the range of 20 to 30%. When cells are isolated from the tissue section, many cells will be missing part of the nucleus. It is also conceivable that the primers employed in the experiment may fail to detect the V gene of a given cell, either because the V gene of that cell is not known yet or because a mutation occurred in the primer binding regions.