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→Workshop 1 (Molecular/DNA cloning)
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*11. 3 centrifuge cycles with different buffer; the last one with water in order to have dna in the final sample | *11. 3 centrifuge cycles with different buffer; the last one with water in order to have dna in the final sample | ||
*12. polymerase master mixer (half of the total mix; 15ul; from one dna makes more dna) + sample (10ul) + primer forward (2,5ul) + primer reverse (2,5ul) | *12. polymerase master mixer (half of the total mix; 15ul; from one dna makes more dna) + sample (10ul) + primer forward (2,5ul) + primer reverse (2,5ul) | ||
*PCR multiplication HOWTO: | |||
a) We start at 95C two strands divides, hydrogen bonds will break at this temperature; DNA is accessible | |||
<br>b) Annealing: primers bind to the targets Roughly at the 65C | |||
<br>c) 72C polymerase start elongation of dna. Copies into one direction. It is called TAQ polymerase; comes from specific species called /thermus aquaticus/; lives in gazers and very stable; For every 1000 base pairs you need to copy we need around 60s. | |||
<gallery> | <gallery> | ||