enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from dna building blocks, the nucleotides, by using single-stranded dNA as a template and dna oligonucleotides(also called DNA primers), which are required for initiation of dna synthes V Agarose gel electrophoresis Parkinson Disease: Point mutation, Parkin( PARK 2)exon4, AGC - AAC PCR 2. Agarose gel electrophoresis is a method to separate DNA, or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Shorter molecules move faster and migrate farther than longer ones 3. Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting 2. Visualisation: Ethidium Bromide(EtBr)and dyes The most common dye used to make dna or rna bands visible for agarose gel electrophoresis is ethidium bromide, usually abbreviated as EtBr. It fluoresces under UV light when intercalated into DNA (or RNA). By running DNA through an EtBr-treated gel and visualizing it with Uv light EtBr is a known mutagen however safer alternatives are 3 Percent agarose Most agarose gels are made with between 0. 7%(good separation or resolution of large 5 lokb dna fragments)and 2%(good resolution for small 0.2-lkb fragments)agarose dissolved in electrophoresis buffer uffers: The most common being: Tris/Acetate/EDTA (TAE 4Steps 1. 1% agarose solution in 20ml TAE. (0.2g agarose in 20ml TAE Carefully bring the solution just to the boil to dissolve the agarose 2. t the solution cool down to about 60 C at room temperature. Stir or swirl the solution while 3.d ethidium bromide stock in the gel solution for a final concentration of 0.5 ug/ml. Be very careful when handling the concentrated stock 4. tir the solution to disperse the ethidium bromide, then pour it into the gel rack Insert the comb at one side of the gel, about 5-10 mm from the end of the gel 5. en the gel has cooled down and become solid, carefully remove the comb. The holes that remain in the gel are the wells or slots 6. ut the gel, together with the rack, into a tank with TAE. The gel must be completely covered with TAE. with the slots at the end electrode that will have the negative current 5.Analysis After the gel has been prepared, use a micropipette to inject about 3ul of stained dnA (a dNA ladder is also highly recommended). Close the lid of the electrophoresis chamber and applyenzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. V Agarose gel electrophoresis Parkinson Disease: Point mutation, Parkin(PARK2) exon4, AGC – AAC 1.Purpose: 1.Genomic DNA extraction - PCR - Agarose gel electrophoresis -Sequence 2.Agarose gel electrophoresis is a method to separate DNA, or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Shorter molecules move faster and migrate farther than longer ones . 3.Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting 2.Visualisation: Ethidium Bromide (EtBr) and dyes The most common dye used to make DNA or RNA bands visible for agarose gel electrophoresis is ethidium bromide, usually abbreviated as EtBr. It fluoresces under UV light when intercalated into DNA (or RNA). By running DNA through an EtBr-treated gel and visualizing it with UV light. EtBr is a known mutagen, however, safer alternatives are available. 3.Percent agarose Most agarose gels are made with between 0.7% (good separation or resolution of large 5– 10kb DNA fragments) and 2% (good resolution for small 0.2–1kb fragments) agarose dissolved in electrophoresis buffer. Buffers:The most common being: Tris/Acetate/EDTA (TAE) 4.Steps 1. 1% agarose solution in 20ml TAE. （0.2g agarose in 20ml TAE ） Carefully bring the solution just to the boil to dissolve the agarose. 2.t the solution cool down to about 60 °C at room temperature. Stir or swirl the solution while cooling. 3.d ethidium bromide stock in the gel solution for a final concentration of 0.5 ug/ml. Be very careful when handling the concentrated stock. 4.tir the solution to disperse the ethidium bromide, then pour it into the gel rack. Insert the comb at one side of the gel, about 5-10 mm from the end of the gel. 5.en the gel has cooled down and become solid, carefully remove the comb. The holes that remain in the gel are the wells or slots. 6.ut the gel, together with the rack, into a tank with TAE. The gel must be completely covered with TAE, with the slots at the end electrode that will have the negative current. 5..Analysis After the gel has been prepared, use a micropipette to inject about 3µl of stained DNA (a DNA ladder is also highly recommended). Close the lid of the electrophoresis chamber and apply