https://wiki.xenbase.org/xenwiki/index.php?title=Extraction_of_DNA_from_Single_Embryos_(Sive_et_al._2000)&feed=atom&action=historyExtraction of DNA from Single Embryos (Sive et al. 2000) - Revision history2024-03-29T10:43:17ZRevision history for this page on the wikiMediaWiki 1.40.1https://wiki.xenbase.org/xenwiki/index.php?title=Extraction_of_DNA_from_Single_Embryos_(Sive_et_al._2000)&diff=47396&oldid=previmported>Virgilio: Created page with "'''Information adapted from:''' ''Early Development of Xenopus Laevis: A Laboratory Manual.''[http://www.xenbase.org/literature/book.do?method=display&bookId=22] 2000; (First e..."2011-12-19T16:39:48Z<p>Created page with "'''Information adapted from:''' ''Early Development of Xenopus Laevis: A Laboratory Manual.''[http://www.xenbase.org/literature/book.do?method=display&bookId=22] 2000; (First e..."</p>
<p><b>New page</b></p><div>'''Information adapted from:'''<br />
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''Early Development of Xenopus Laevis: A Laboratory Manual.''[http://www.xenbase.org/literature/book.do?method=display&bookId=22]<br />
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2000; (First ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory Press. <br />
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Sive, Hazel (Author), Grainger, Robert M (Author), and Harland, Richard M (Author).<br />
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'''DNA Isolation'''<br />
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Genomic DNA is used for Southern blotting, for determining gene structure, and for detecting the presence or absence of genes of interest.<br />
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'''Extraction of DNA from Single Embryos'''<br />
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1. Homogenize the embryo using a micropestle in a microcentrifuge tube, containing 0.5 ml of homogenization buffer (see [http://www.xenbase.org/xenwiki/index.php/DNA/RNA_preparation]).<br />
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2. Store the homogenate at -20 deg C until ready to process.<br />
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3. Thaw the homogenate and add 2.5 ul of proteinase K 920 mg/ml).<br />
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4. Mix well and incubate overnight at 55 deg C.<br />
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5. Extract with 1 volume of aqueous phenol.<br />
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Note: If the DNA isolation is viscous and seems to drag phenol with it, it may be easier to remove the lower phenol layer rather than removing the upper aqueous layer.<br />
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6. Extract once with a 1:1 mix of phenol:chloroform, and then once with chloroform.<br />
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Note: Embryos appear to contain compounds that inhibit restriction digestion. It is essential to thoroughly extract the DNA in order to remove these inhibitors.<br />
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7. Transfer the aqueous phase to a fresh tube and precipitate the DNA by adding ammonium acetate to 2 M and 0.6 volumes of isopropanol.<br />
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8. Mix gently but thoroughly by inverting the tube.<br />
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Note: The precipitate may be viscous and mixing can take some time.<br />
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9. Hold the tube on ice until a stringy DNA precipitate appears (~30 minutes). This may not be apparent from a single embryo before stage 20-30.<br />
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Note: If DNA fails to precipitate on ice, store at -20 deg C overnight.<br />
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10. Recover the precipitate by centrifuging at 12,000xg for 5 minutes.<br />
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11. Wash the pellet in 70% ethanol. Redissolve in 10 ul of TE.<br />
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12. Add RNase A to 10 ug/mo and RNase T1 to 10 ug/ml. Incubate for 30 minutes at room temperature.<br />
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13. Precipitate the reaction by adding ammonium acetate to 2 M and 0.6 volumes of isopropanol.<br />
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14. Centrifuge at 12,000xg for 5 minutes and resuspend the pellet in 20 ul of TE<br />
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Note: The older the embryo, the greater number of nuclei and the greater the amount of genomic DNA.</div>imported>Virgilio