BIO-RAD Lambda DNA Kit, and BIO-RAD Forensic DNA Fingerprinting Kit Section Concepts: DNA Scissors Activity, restriction enzymes, gel electrophoresis,

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BIO-RAD Lambda DNA Kit, and BIO-RAD Forensic DNA Fingerprinting Kit Section Concepts: DNA Scissors Activity, restriction enzymes, gel electrophoresis, standard curve

Background Information Restriction enzymes: found in bacteria and archaea, are enzymes that cuts DNA at specific recognition nucleotide sequences known as restriction sites. Gel Electrophoresis: a method used in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments, or to separate proteins by charge. Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose matrix.

Restriction Enzymes + Gel Electrophoresis Restriction enzymes video Gel Electrophoresis animation

Timeline/Overview Monday: Lecture Tuesday: pre-lab quiz, lambda DNA (run gel and visualize) Thursday: DNA fingerprint (restriction digest), Analyze lab (work on packets) Monday: No school Tuesday: DNA fingerprinting kit (run gel, visualize) Thursday: Packet due, post quiz

Necessary Preparation for Lab Lambda Before Lambda Lab Label tubes (pg 17) Aliquot DNA & LD, heat Pour 8 Gels (10 well comb, 1%=1g/100ml, add EtBr) Prepare TAE

Necessary Preparation for Lab DNA Finger printing DNA fingerprinting Labels 8—clear 1.5mL tubes ENZ (enzyme) 8—clear 1.5mL tubes M (marker) 8—clear 1.5mL tubes LD (loading dye) 8--green 1.5mL tubes CS 8—blue 1.5mL tubes S1 8—orange1.5mL tubes S2 8—violet 1.5mL tubes S3 8—red 1.5mL tubes S4 8—yellow 1.5mL tubes S5 Before Digest Set water bath for 37C Rehydrate & aliquot (page 13) EcoRI/PstI enzyme mix (12 hours before use) Crime scene DNA Suspects 1-5 DNA Before Running Gel Prepare electrophoresis TAE buffer 11/13 at before 5 th period Pour 8 gels (1%, EtBr, 10 well comb) Prep. HindIII standard (right before class) Aliquot HindIII (marker) Loading dye

Lambda DNA tips Draw gel to before loading Carefully load Run at 130volts

DNA Fingerprinting Lab - Background Information DNA Fingerprinting: technique used by scientists to identify an individual based on their specific DNA profile Restriction Digestion of DNA: restriction enzymes recognize particular sequences in a segment of DNA and cuts the DNA molecule at that point Plasmids: circular, non- chromosomal pieces of DNA that can replicate in and are commonly found in bacteria

DNA Fingerprinting DNA Fingerprinting Video

Plasmids Restriction Enzyme Digestion of Plasmid DNA Restriction Enzyme Animation Restriction Enzyme EcoR1 Animation

DNA Fingerprinting Tips Place tubes in refrigerator after removing from water bath Gel will be 10 wells, adjust lanes in your protocol to use 2-8