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19.4 Separating and amplifying DNA - Coggle Diagram
19.4 Separating and amplifying DNA
Polymerase chain reaction (PCR)
Ingredients
DNA sample
DNA primer molecules
Free deoxynucleotide triphosphate molecules (dNTPs) to provide energy for formation of phosphodiester bonds
Buffer (pH 7-8)
Taq polymerase
High optimum temperature means higher rate of synthesis during extension stage
High heat tolerance means won't be denatured in denaturation phase and can be reused
Stages
Denaturation
Heat to 95˚C to break H-bonds between DNA strands
Annealing
Cool to about 55˚C to allow DNA primers to bind via formation of H-bonds
Provides binding site for Taq polymerase
Extension
Heat to 72˚C as this is optimum temperature for Taq polymerase
Taq polymerase uses dNTPs to synthesise new DNA strands
Gel electrophoresis
Factors affecting
Net charge of molecules
-ve molecules move towards anode (+ve)
+ve molecules move towards cathode (-ve)
Molecules with more charge move faster
Size of molecules
Small molecules move faster
Gel composition
Size of pores within gel
Separating DNA
DNA is negatively charged so moves towards anode
Size of DNA fragment inversely proportional to distance moved (small fragments move further)
Gel is set and comb is used to create wells for sample loading
Buffer is poured so it covers gel
Micropipette used to deposit samples
Sample containing DNA fragments of known lengths added (ladder)
Buffer poured away, colour stain added, results compared to ladder