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How To Extract DNA From Grapefruit In 5 Minutes
Transcript of How To Extract DNA From Grapefruit In 5 Minutes
DNA from grapefruit
in 5 minutes By Devin Rochussen Materials Water Meat tenderizer Funnel Filter paper Liquid detergent Grapefruit Salt Mortar & pestle Isopropyl alcohol Large test tube Ice Procedure References Austin Community College District - Start Here. Get There [Internet]. Austin (TX): Austin Community College
[ACC]; [cited 2011 April 21]. Available from: docs.google.com/viewer?a=v&q=cache:d6F1gMqcwIEJ:www.austincc.edu/biotech/QualityAssurance/SOP_DNA%2520Extraction_instructor_copy.doc+DNA+extraction&hl=en&pid=bl&srcid=ADGEESjHH6kaoyng81- 6MJmU3hsdrh5sabdpf514ViO-vyK1m5PTpSUlSYL-G9d6HWZRxyXjBS8EbLLebTshBUr6pn9MvGUD9nv-
Learn.Genetics [Internet]. 2011. Salt Lake City (UT): The Genetic Science Learning Centre [uUtah]; [cited 2011
April 21]. Available from: learn.genetics.utah.edu/content/labs/extraction/howto/faq.html
TERPConnect [Internet]. [Date of publication unknown]. College Park (MD): University of Maryland [UMD]; [cited
2011 April 21]. Available from: terpconnect.umd.edu/~nsw/ench485/lab2.htm
The Gene School [Internet]. [Date of publication unknown]. [Place of publication unknown]: The Gene School;
[cited 2011 April 21]. Available from: library.thinkquest.org/19037/dna_extraction.html
WKU Biology [Internet]. 2005. Bowling Green (KY): Western Kentucky University [WKU]; [cited 2011 April 21].
Available from: bioweb.wku.edu/courses/biol350/GeneCloning5/Review.html Step 1 Why cold, salty, and grinded? Take a two-and-a-half centimetre wide slice from the grapefruit, and remove the peel and skin.
Add the pink pulp to the mortar, along with 10 millilitres (mL) of cold water that has two pinches of salt dissolved in it.
Grind the mixture with the pestle for 10 seconds, or until mixture is homogenous. The mixture should be thick and opaque. The cold water acts to slow down enzymatic reactions, which will contribute to protecting the DNA from enzymes such as DNases and therefore keeps it intact (uUtah 2011). Grinding with the pestle helps break down the cell walls, while the salt provides the DNA with a favourable environment, contributing positively charged ions to neutralize the negatively charged DNA (The Gene School 2011). The salt will also cause the proteins and other cellular debris to clump together later on in the procedure (uUtah 2011). Step 2 Step 3 Step 4 Step 5 Why detergent? Why meat tenderizer? Why alcohol? Why filter? Add 2 mL of liquid detergent to mixture, carefully stirring as to not create any bubbles.
Set aside for 30 seconds. The detergent contains proteases which disrupt and break down the lipid molecules of the cell membrane and nuclear envelope, thus causing the cell to burst open and release its DNA (The Gene School 2011). These enzymes work by breaking down the peptide bonds found in the proteins (UMD 2011). Add a pinch of meat tenderizer to the mixture, and stir gently for 20 seconds.
The mixture should be opaque and pink. The meat tenderizer contains proteases, most commonly bromelain and papain, which work to free DNA by breaking down the histone proteins, figure 1 (uUtah 2011). The papain in meat tenderizer can also work to break down the DNases found in the cytoplasm of the cell, which are the enzymes that would normally consume the DNA, thus preventing the destruction of the DNA (ACC 2011). The mixture needs to be stirred gently, otherwise the meat tenderizer will destroy the DNA along with the proteins. Figure 1: Simplified explanation of the purpose of meat tenderizer (uUtah 2011) Add the mixture to a large test tube via filter paper and a funnel. The filter is used in place of a centrifuge in order to separate the cell scum and debris from the DNA (WKU 2011). Add 5 mL of ice-cold alcohol to the test tube by pouring it down the side slowly, as to create a defined layer of alcohol on top of the solution.
Leave aside for 2-3 minutes, and the DNA will rise to the top of the alcohol layer as a stringy, clear substance with bubbles attached to it.
The stringy substance is actually a mix of DNA and RNA, thus making this lab experiment a nucleic acid extraction, rather than just a DNA extraction (uUtah 2011). DNA will not dissolve in alcohol, so the DNA precipitates. It is less dense than water, so it floats up into the alcohol layer (The Gene School 2011). The alcohol is kept cold to allow the DNA to precipitate more quickly (uUtah 2011).