Team:Exeter/lab book/proto/7

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Protocol 7


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

Two-Step PCR
Make-up a 50µL total solution in a PCR tube of: 5µL of 10ng/µL genomic DNA template 1µL dNTPs 1µL Forward Primer (@100pmol/µL) 1µL Reverse Primer (@100pmol/µL) 10µL High-Fidelity Phusion 5x Buffer 1µL High-Fidelity Phusion polymerase 31µL MilliQ H2O Run a two-step PCR experiment using the following settings: Stage 1: 98oC for 30 seconds for 1 cycle Stage 2: 98oC for 10 seconds followed by 72oC for 20 seconds for 30 cycles Stage 3: 72oC for 5 minutes for 1 cycle

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