PCR The polymerase chain reaction Crick and Watson

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PCR The polymerase chain reaction

PCR The polymerase chain reaction

Crick and Watson – structure of DNA

Crick and Watson – structure of DNA

A double helix with two antiparallel strands

A double helix with two antiparallel strands

“It has not escaped our notice that the specific pairing we have postulated immediately

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a copying mechanism for the genetic material. ”

5’ end Ends 3’ end 3’end 5’ end

5’ end Ends 3’ end 3’end 5’ end

My gene: 5’TCACGACGTTGTAAAACGACGTCTATACCGAGCTCCTCTCTCATAGCTGTTTCCTGTGTGAA 3’ I design custom oligonucleotides to the “sense” strand at the

My gene: 5’TCACGACGTTGTAAAACGACGTCTATACCGAGCTCCTCTCTCATAGCTGTTTCCTGTGTGAA 3’ I design custom oligonucleotides to the “sense” strand at the beginning and the “antisense” at the end Sense 5’TCACGACGTTGTAAAACGACGTCTATACCGAGCTCCTCTCTCATAGCTGTTTCCTGTGTGAA 3’ Anti 3’AGTGCTGCAACATTTTGCTGCAGATATGGCTCGAGGAGAGAGTATCGACAAAGGACACACTT 5’ “forward primer” 5’TCACGACGTTGTAAAACGACG 3’ “reverse primer” 3’GTATCGACAAAGGACACACTT 5’

http: //www. people. virginia. edu/~rjh 9 u/pcranim. html

http: //www. people. virginia. edu/~rjh 9 u/pcranim. html

DNA hybridization rate is altered by changing Temperature: template dependent Salt Concentration: can be

DNA hybridization rate is altered by changing Temperature: template dependent Salt Concentration: can be standardized DNA concentration: can be standardized Time: can be standardized

Thermal cycler Heated lid (prevents evaporation problems) 96 well plate format Peltier effect heating

Thermal cycler Heated lid (prevents evaporation problems) 96 well plate format Peltier effect heating / cooling block Microprocessor controlled thermal program

Real-time PCR Quantitative Fast High sample processing speed Very sensitive

Real-time PCR Quantitative Fast High sample processing speed Very sensitive

Parasitic Worm Inoculation Levels Violet = 200 worms per root Red = 50 worms

Parasitic Worm Inoculation Levels Violet = 200 worms per root Red = 50 worms per root Blue = 20 worms per root

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