Team:Exeter/lab book/proto/5

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Protocol 5


Protocols \| Single Gene Plasmids and Enzyme Characterisation \| Showcasing Polysaccharide Production \| The 3-Gene Inducible Plasmid Operon Construction \| Glycobase

Annealing Oligomer Primers
A variation of the oligonucleotide annealing protocol from Bioneer was used: Mix the concentrated complementary oligonucleotides together at 1:1 molar ratio in a micro centrifuge tube. Add to each primer tube annealing buffer calculated to achieve 200uM, e.g.10 mM Tris, 0.1 mM EDTA, 50 mM NaCl (pH 8.0). Mix 25ul of each appropriate oligomer pair together in a PCR tube. Incubate the oligonucleotides at 95°C for 2 minutes. Decrease temperature by 0.5°C every thirty seconds for 130 cycles until room temperature is reached Heat at 95° for two minutes then cool to 30° over an hour by decreasing by 0.5°C per thirty second cycle for the 130 cycles. Cool rapidly to 4° or place in the freezer.