By David M. Terrian
A suite of comfortably reproducible experimental equipment for learning the subversion of ordinary telephone signaling that results in melanoma. awarded in step by step element to make sure winning effects, the tools contain confirmed suggestions for the research of apoptosis and mobile loss of life, complementary protocols for manipulating and/or tracking oncogenic signs in melanoma cells, and methods for learning protein-protein interactions. a close protocol for shooting natural samples of malignant cells from frozen tissue specimens and substitute suggestions for reading their genomic DNA can also be defined.
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Additional info for Cancer Cell Signaling: Methods and Protocols (Methods in Molecular Biology Vol 218)
There are two alternative and easier methods available now for cloning genomic DNAs. BAC library DNA is commercially available (ResGen). The gene of interest can be screened using PCR, and the BAC plasmid containing the genomic DNA fragment can be purchased. Further, the genomic DNA can be directly PCR-amplified using sequence data available from mouse genome project (Celera). 6. Homozygous gene targeting can also be achieved by a consecutive targeting using a construct carrying another selection marker (31).
25 g. 2. 05 mL. 3. PBS(-), 500 mL (see Note 3). 4. Gelatin 1. 5 g of gelatin (Sigma G1890) in 500 mL PBS(-), autoclave, and keep at 4ºC. 2. Cover the culture plates with gelatin solution for 20 min at room temperature and remove it before plating STO or ES cells. 5. STO Cell Culture 1. Culture STO cells in ES cell medium without LIF in gelatinized flask. 2. Treat confluent STO cells with 6000–10,000 rads of gamma irradiation before plating ES cells (see Note 4). 6. 2X Cell Stock Solution 1. 20% dimethyl sulfoxide (DMSO).
Transfer cells into T-25 flask with feeder cells and culture at 37ºC, 5% CO2. Change medium at d 1 or d 2 and passage at d 3. 8. Passage of ES Cells 1. 2. 3. 4. 5. 6. Discard medium and wash once with 5 mL of PBS(-). 5 mL of Trypsin/EDTA and sit for 2–3 min at room temperature. Tap the flask to remove cells. Add 5 mL of medium and pipeting. Transfer cells into T-75 flask with feeder cells and add medium to 15 mL. Change medium at d 1 or d 2 and passage at d 3. 2. 1. Cloning of Genomic DNA Coding a Target Gene A genomic DNA coding the gene of interest for constructing a targeting vector is needed.
Cancer Cell Signaling: Methods and Protocols (Methods in Molecular Biology Vol 218) by David M. Terrian