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Lab4_09_ - Author Jack Reifert Page 1 Week 4(Oct 19-23...

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Author: Jack Reifert Page: 1 Week: 4 (Oct 19-23) Laboratory #4 Part Ia) Preparing the Agarose Gel to Visualize the DNA of the Restriction Digest From Last Lab: (2 groups will share 1 gel) Take Home Point: DNA separation by filtration through a matrix. Meaning the DNA is separated by its size by “forcing” the DNA strands through tiny pores formed by the agarose. This is a common theme for separating many biological samples: proteins, DNA, RNA, etc…. (Size serves as a useful tool for analysis) Other Points: - Structure of agarose - Use of buffers and what purpose they serve Basic Steps: 1) Make a 0.7% solution of Agarose in TBE buffer (See example calculation below) 2) Heat the agarose to make a solution 3) Allow the agarose to cool and pour your gel carefully - Be sure to consider the following; - The quality of the gel depends on it setting peacefully until solid - The comb is used to make “wells” where you will load your samples. These wells don’t do much good if they go all the way through the gel – that will cause your samples to fall out the bottom. Make sure there is space between the gel and the bottom of the casting tray! - The wells should also not be against the back of the gel for the same reason - Make sure the gel caster is level for a level gel 4) Allow the gel to cool and solidify in the casting tray for 30 minutes Example Calculation for % solution: - A percent solution comes from water: 1 mL of water weighs 1 gram (nifty eh?) - Therefore, this is considered 100% and this is used for any mix of solids and liquids - 1 gram in 1 ml would be 100% SO here it goes: 40mL of buffer @ 100% would be = 40 grams 40mL of buffer @ 10% would be = 4 grams (40 x 0.1 = 4) 40mL of buffer @ 1% would be = 0.4 grams (40 x 0.01 = 0.4) And finally, 40mL of buffer @ 0.7% would be 0.28 grams (40 x 0.007 = 0.28) Part Ib) Gel Electrophoresis: Take Home Point: Separation of DNA by size by “forcing” the DNA with electricity through an agarose matrix.
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