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DNA Technology

DNA Technology. Restriction Digests and Gel electrophoresis. DNA Extraction (taking it out). Collect DNA sample (blood, saliva, hair follicle, skin) Burst open the cells using lysis buffer and free the DNA (like extraction lab). Microsatellite regions.

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DNA Technology

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  1. DNA Technology Restriction Digests and Gel electrophoresis

  2. DNA Extraction (taking it out) • Collect DNA sample (blood, saliva, hair follicle, skin) • Burst open the cells using lysis buffer and free the DNA (like extraction lab)

  3. Microsatellite regions • Almost all DNA between humans is identical (99.9%), except in non-protein coding sites called microsatellite regions • Where we look when comparing DNA to solve crimes or for paternity

  4. DNA Amplification • PCR=Polymerase chain reaction • Makes identical copies of the portion of the DNA

  5. Restriction Digest • Now cut up the DNA based on its base pair sequence • We use restriction enzymes from bacteria. They cut at very specific sequences. Example: BsuRI GGCC going 5’ to 3’ • Why would bacteria need enzymes to cut up DNA?

  6. Practice • Where would BsuRI cut for the following individuals and how many pieces would result? • Person 1 5’TGGCCATGGCGGCC3’ 3’ACCGGTACCGCCGG5’ • Person 2 5’TTCCGGCCTCCAGA3’ 3’AAGGCCGGAGGTCT5’

  7. Volume • We will be measuring tiny volumes-microliters written µL. • There are 1000 µL in I mL. There are 1000mL in 1 L. So 1 µL is 1,000,000 of a liter=very small. • We use micropipettes • Use p20- can measure between 2 and 20 µL accurately • Top number = tens place, middle= ones place, and bottom=tenths place

  8. Micropipettes • Very expensive-$300 each • Liquid must never touch the barrel, it must always be in a new tip • Always hold the micropipet vertically • Work at eye level

  9. Practice volumes • How would you dial to 6.5 µL? • How would you dial to 20 µL? • How would you dial to 2 µL?

  10. Diagram • Draw pipette diagram on board

  11. Micropipet Use 1. Dial to correct volume and lock plunger 2. Put on fresh tip 3. Push plunger down until first stop=resistance 4. Insert tip into liquid and release plunger SLOWLY 5. Place tip into container to move liquid to 6. Push down to the 2nd stop. Remove micropipet BEFORE you release plunger

  12. Gel Electrophoresis • A process used to separate our cut DNA by LENGTH! • Gel is a meshwork of fibers-agarose • DNA is NEGATIVE • What attracts a negative? • Negative at the top of the gel, positive at the bottom • Electric current pulls DNA through the gel • Use a mutagen called ethidium bromide to stain • Which pieces will move faster if it is like the game?

  13. Activity • The restriction enzyme you will use will cut at 5’ to 3’ GG CC, between the Gs and Cs • Draw where you would cut the sequences and draw a line where they would move to on the gel • Which person committed the crime?

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