DNA Extraction from Archival Formalin-fixed, Paraffin-embedded Tissue Sections
	 
	
		
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					DNA Extraction from Archival Formalin-fixed, Paraffin-embedded Tissue Sections | 
		
			| Author: Shi et al. | 
		
			| Source: Contributed by APostodoc | 
		
			| Abstract: Describes two methods of extracting DNA from archived, paraffin-embedded sections. One is the standard non-heating enzymatic digestion method; the other is heating method based on the principles of the antigen retrieval technique, which yields better DNA quantity. | 
		
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					Section 1. Non-heating DNA Extraction Protocol  
					
						
							Cut paraffin block at 10 µm and collected in  an autoclaved plastic microtube (1.5 ml).
						
							Add 1 ml xylene to the microtube containing tissue sections for 30 min for two changes, 
						
							Add 100% and 75% ethanol for 30 min with two changes
						
							Wash with PBS for 15 min with two changes 
						
							Add 500 µl of lysis buffer (proteinase K 20 mg/ml, 50 µl, 1 M Tris-HCl solution 10 µl, 0.5 M EDTA 2 µl, 10% SDS 100 µl, and distilled water 838 ml) 
						
							Incubated at 52°  C overnight until all tissue fragments were dissolved completely
						
							Add 500 µl phenol:chloroform:isopropanol alcohol at 25:24:1 to the de-waxed tissue
						
							Mix by vortex 
						
							Centrifugation at RT, 12,000 x g for 10 min 
						
							Transfer the supernatant fluid to an autoclaved microtube using a 100-µl pipette
						
							Add one volume of chloroform to the supernatant, mixed by vortexing
						
							Centrifuged at 12,000 x g for 5 min. 
						
							Carefully remove the upper aqueous supernatant to another fresh microtube
						
							Adding 0.1 volume of 3 M sodium acetate to the new tube
						
							Mix by vortexing
						
							Add 1 volume of isopropanol, and incubate at -20C overnight. 
						
							The precipitated DNA was centrifuged at 12,000 x g at 4C. 
						
							Discard the supernatant fluid and wash once with 75% ethanol. 
						
							Collect the extracted DNA after further centrifugation. 
						
							Dissolve the final yield of DNA in 50 µl distilled water after drying completely in a hood.  
					  
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	Section 2. Heating Protocol for DNA Extraction 
	 
	Overview 
	Materials and Reagents  
	Procedure  
	  
	 
	Reference  
	Shi et al, Journal of Histochemistry and Cytochemistry, Vol. 50, 1005-1011, August 2002. 
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