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PCR (Ingrediants: (dNTPs, Reaction buffer, Forward primer, Reverse primer,…
PCR
Ingrediants:
dNTPs
Reaction buffer
Forward primer
Reverse primer
Taq polymerase
Template DNA
dH2O
Function:
Amplify DNA for cloning
Amplify DNA for sequencing
Detections of mutations
Detection of relatedness
Steps:
Heat to 94C to separate DNA strands
Cool to 50-60C to allow premieres to anneal
Elongate strands at 72C
PCR machine
Digitally controls temperature
Reactions performed in small tubes
Finishes when Taq runs out
Reverse transcription PCR functions:
Allows detection of gene expression
Make cDNA
Virus testing
DNA polymerase
Taq
From Thermus aquaticus bacterium
Found naturally in hot springs and deep sea vents
Very good at replicating DNA at high temp
AMPLICON
Region of single stranded DNA to be amplified
Primers:
Designing
18-22 nuleotides in length
GC content of around 40% - 60%
Melting temp of 55-65C
Both primers impair should have a similar Tm
Must be specific
Analysing products
Gel elecrophoresis
Agarose gel
Product + fluorescent dye
Smaller products move faster
Intensity of band can show amount