| gab9281 | Дата: Четверг, 05.12.2013, 15:08 | Сообщение # 1 |
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The standard CGH protocol relies on the availability of macroscopic tumor samples, which do not contain high proportions of nontumor normal cells. Most tumor samples are contaminated with such normal tissue and if the ratio of tumor: normal cells is lower than 1: 1 (35), CGH-analysis does not provide reliable results. In such cases, a combination of microdissection tumor tissue, DOP-PCR, and CGH can be the solution. As shown by Kuukasjarvi and coworkers (36) this approach provides an excellent tool to study genetic aberrations in different histological subpopulations of malignant, as well as, precancerous, lesions. DOP-PCR CGH analysis also improves the success rate of conventional paraffin-block CGH (36), as the poor-quality low-yield DNA can be compensated for by universal DNA amplification using DOP-PCR. 1. For each slide to be hybridized, add 1.5 |J.L each of biotin and digoxigenin labeled chromosome specific probes (ONCOR, Inc.) and 3 |jL of 1 |J.g/|JL COT1-DNA (Gibco) to 20 |jL of the hybridization buffer in a 1.5-ml microtube, vortex, and spin down (see Note 10). Figure 4.10 Population Distribution of Zambia by Age and Sex, 2000 http://nokeratosis.com - Purchase imiquimod cream 5% no rx • New laboratory test. 1. Wash the cells twice at room temperature by adding 6 mL PBS per dish, swirling dishes, and pouring off. http://cusreview.com/article/cialisandpenissize.php - cialis increase penis size 4. If the SSCP pattern definitely appears abnormal, but the sequence is normal, then recheck the percentage of tumor cells in the tissue. If the tumor percentage is low (<25%), then the investigator may need to cut out the abnormal band from the SSCP gel, extract the DNA and reamplify (nested primers work best in this setting). If the tumor percentage is adequate, then the investigator needs to use new primers to increase the fragment size because the base change lies too near the primer to detect with the sequencing approach above. Alternatively, the fragment can be cloned with sequence done off an universal site in the vector that is adjacent to the primer sequence. 2. The shiny side of the polycarbonate membrane is placed so that it sticks onto the parafilm (dull side up). Mild pressure is exerted by the gloved fingertips to accomplish this step. All four sides of the filter are then secured to the glass plate with Scotch tape (see Note 7).
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| Nuuxazgok | Дата: Суббота, 03.06.2017, 21:44 | Сообщение # 2 |
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