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DNA Extraction and Gels

DNA Extraction and Gels. Manipulation of DNA. After discovering DNA was the carrier of genetic info, it became apparent that control over its mechanisms would be essential

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DNA Extraction and Gels

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  1. DNA Extraction and Gels

  2. Manipulation of DNA • After discovering DNA was the carrier of genetic info, it became apparent that control over its mechanisms would be essential • We have ways of isolating DNA, determining the order of its nucleotides, and can even insert of piece of DNA from one organism to another

  3. DNA Extraction • To get at DNA, we have to remove the two membranes that protect it • Cell membrane and nuclear envelope • These are made of phospholipid bilayers • Detergents are used to dissolve the lipids to expose the genetic material • Next, a protease is used to dissolve unwanted proteins • Sodium acetate further precipitates the remaining protein

  4. Next, the sample is centrifuged, and the protein can be removed • DNA can be precipitated by the addition of cold ethanol • This both precipitates the DNA and washes the salt • Finally, the solution is centrifuged, and the pellet of DNA is removed

  5. Cutting the DNA • The DNA extracted is often very long • Too long to be useful in many cases • Restriction enzymes are used to cut the DNA • However, it is not a random cut site, rather a very specific sequence • There are many different enzymes (~1000), many with their own unique sequence they attach to • They do not cleave the DNA straight down, instead the cut the bond between adjacent nucleotides

  6. The uneven cutting creates what is known as “sticky ends” • They are “sticky” because they are single stranded • DNA is normally doubled stranded, and naturally anneals (reforms hydrogen bonds) into double • There is a random number of cut sites on every strand of DNA • But, in the end, the sample is cut into smaller pieces

  7. Gel Electrophoresis • These smaller segments can be run through a gel • The gel is a semi-solid, porous medium that DNA can move through • Electrodes are set at either end to make one positive, the other negative • DNA is slightly negative, and will be attracted to the positive end • The smaller samples can move through the gel faster, and will make it farther down

  8. Each band represents a strand of DNA, of a specific length • The bands furthest down are the smallest • Gels are normally run to compare a sample of DNA to a known sample, cut with the same enzymes • If the bands line up, it is likely that the samples are the same • This is very useful in DNA fingerprinting

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