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PCR & Gel Electrophoresis - Chaidir Adam 2018
- 1. 2018 Department of Biology Education Postgraduate Program Palangkaraya University PCRPCR IN VITRO GENE MULTIPLICATION Polymerase Chain Reaction AND GENE VISUALIZATIONGEL ELECTROPHORESIS CHAIDIR ADAM ACD217004
- 2. INTRODUCTIO N BIOLOGY MOLECULAR Interdisciplinary Science Biochemistr y Cell Biology Genetics Includes study MOLECULAR biological activities on level has a role Evolutionary BIOLOGY
- 3. INTRODUCTIO N BIOLOGY MOLECULAR Mengalami perkembangan yang sangat pesat didukung dengan perkembangan teknologi PCRPolymerase Chain Reaction gene multiplication 2 GEL ELECTROPHORESIS gene visualization 1
- 4. PCR Polymerase Chain Reaction a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA we can make many copies (millions or billions) of a particular region of DNA
- 5. PCR Polymerase Chain Reaction DNA SAMPLES as a template Plasmid DNA
- 6. PCR Polymerase Chain Reaction PRIMERS short nucleic acid sequence that provides a starting point for DNA synthesis PRIMERS DESIGN based on known DNA sequence from GeneBank Database PRIMERS LENGTH 18 – 22 bp (less than 18 bp can lead into mispriming possibility)
- 7. PCR Polymerase Chain Reaction NUCLEOTIDES DNA building blocks deoxyadenosine triphosphate deoxyguanosine triphosphate deoxythymidine triphosphate deoxycytidine triphosphate
- 8. PCR Polymerase Chain Reaction PCR BUFFER MgCl2 Maintain medium pH polymerase cofactor
- 9. PCR Polymerase Chain Reaction Thermus aquaticus Taq Polymerase Heat resistant enzyme that isolated from Thermus aquaticus other thermal-stable DNA polymerases isolated from other thermophilic species: Pyrococcus furiosus (Pfu Polymerase), Thermus thermophilus (Tth Polymerase), Thermus flavus (Tfl Polymerase), Thermococcus litoralis (Tli Polymerase aka Vent Polymerase), and Pyrococcus species GB-D (Deep Vent Polymerase)
- 10. PCR Polymerase Chain Reaction THE PROCESS 1.Denaturatio n 2.Annealing 3.Extension
- 11. GEL Electrophoresis DNA is negatively chargewhen placed in an electrical field, DNA will migrate toward the positive pole (anode). An agarose gel is used to slow the movement of DNA and separate by
- 16. GEL Electrophoresis STAIN THE RESULT 1. Ethidium bromide is a powerful mutagen and is moderately toxic 2. Be safe!!! Put on masks and gloves
- 17. GEL Electrophoresis Ethidium Bromide safer alternatives to 1.Methylene Blue 2.BioRAD – Bio-safe DNA Stain 3.Ward’s – Quickview DNA Stain 4.others ADVANTAGES 1. Inexpensive 2. Less toxic 3. No UV light required 4. No Hazardous waste disposal DISADVANTAGES 1. Less sensitive 2. More DNA needed on gel 3. Longer staining/destainin time
- 19. 2018 Department of Biology Education Postgraduate Program Palangkaraya University THANK YOU DISCUSSIO N CHAIDIR ADAM ACD217004