Team:Exeter/lab book/proto/9

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Protocol 9


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

PCR purification using QIAquick
Add 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix. Check that the colour of the mixture is yellow. If the colour of the mixture is orange or violet, add 10µL of 3M sodium acetate, pH 5.0, and mix. The colour of the mixtue will turn to yellow. Place a QIAquick spin column in a provided 2mL collection tube. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30-60 seconds. Discard flow-through. Place the QIAquick column back into the same tube. Centrifuge the column for an additional minute. Place QIAquick column in a clean 1.5mL collection tube. To elute DNA, add 50µL Super Pure H2O to the center of the QIAquick membrane and centrifuge the column for 1 minute. Alternatively, for increased DNA concentration, add 30µL Super Pure H2O to the center of the QIAquick membrane, let the column stand for 1 minute, and then centrifuge. If the purified DNA is to be analysed on a gel, add 1 volume of loading dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

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