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ES Cell Culture and Manipulation(2)
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When cells are confluent and media is good and yellow scrape cells to make DNA. Aspirate media and carefully rinse each well 2 times with lml of incomplete PBS. Add another lml PBS and scrape cells with a rubber policeman, transfer to an eppendorf tube and freeze at -20°C until ready to extract DNA.
Gelatin treatment of Tissue-culture plates
Make a 0.1% solution of gelatin (Sigma # G-1890) in Milli-Q water and autoclave, cool. Add enough gelatin to each plate, or well of a plate, to cover the bottom and let sit at least 5 minAspirate and add STO/SNL media.
Extraction of DNA from ES cells
Rapid Preparation of DNA from ES cells in 24-well tissue culture dishes
This simple method based on a protocol described by Miller et al. (1988) involves salting out cellular proteins with a saturated NaCl solution. It does not require extraction with phenol. Sufficient DNA can be obtained by this method for screening by Southern blot analysis (3-5 g)
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