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•Polymerase Chain Reaction (DNA Polymerase – duplicates DNA when cells divide) •DNA copying machine – creates the compliment strand (ATCG-TAGC) •PCR is used to amplify a short, well-defined part of a DNA strand. This can be a single gene, or just a part of a gene •Requires certain components: DNA template, contains the region of the DNA fragment to be amplified Two primers, determines beginning and end of the region to be amplified DNA-Polymerase, which copies the region to be amplified Nucleotides, from which the DNA-Polymerase builds the new DNA Buffer, chemical environment for the DNA-Polymerase Thermocycler – amplification machine •detection of hereditary diseases •the id of genetic fingerprints •the cloning of genes •paternity testing. *Note: Each gel chamber holds 25 ml of the Agarose solution.* 1. Insert the comb and then tape ends of gels with scotch tape. Label the tape with your name. 2. Place gel tray into a clean weigh boat to contain spills. 3. Add 22.5 ml of dH20 and 0.25g of Agarose to a 125 ml Erlenmeyer flask. 4. Microwave until it turns clear and all Agarose is in solution. (roughly 40 seconds-CAUTION: It bubbles quickly so do 10 second intervals) 5. Add 2.5 ml of 10x TAE buffer, then add 20 ml ethidium bromide (EtBr). 6. Gently pour solution into gel tray, remove bubbles and let it sit for 20 minutes. Agarose gel electrophoresis is a procedure that consists of injecting DNA into agarose gel and then applying an electric current to the gel. As a result, the smaller DNA strands move faster than the larger strands through the gel toward the positive current. The size of the PCR product can be determined by comparing it with a DNA ladder, which contains DNA fragments of known size, also within the gel Our “dye” (EtBr) is added before -Fill chamber with 2500 ml of 1x TAE -Put gel tray into chamber -Add dye to the extracted/digested DNA -dye weighs the DNA (dye + glycerol) -Load 1st well with 15ml DNA Marker -Load remaining wells with 15ml dye/DNA mix Gene Ruler 1kb Smaller fragments migrate faster DNA Is (+) 3 kb DNA Fragment