Team:Exeter/lab book/proto/12

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Revision as of 00:54, 25 September 2012

Protocol 12


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

3-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 three-step PCR experiment using the following settings:/p> Stage 1: 98^oC for 5 minutes for 1 cycle Stage 2: 98^oC for 30 seconds followed by 65^oC for 20 seconds, followed by 72^oC for 30 seconds, all for 30 cycles Stage 3: 72^oC for 5 minutes for 1 cycle