Team:Exeter/lab book/proto/8

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Protocol 8


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

Bacterial Genomic DNA Extraction Using GenElute
Pellet 1.5mL of an overnight bacterial broth culture by centrifuging for 2 minutes at 13’000 x g. Remove the culture medium completely and discard. Re-suspend pellet thoroughly in 180µL lysis solution T and add 20µL RNase A solution, mix, and incubate for 2 minutes at room temperature. Add 20µL of Proteinase K solution to the sample. Mix and incubate for 30 minutes at 55oC. Add 200µL of lysis solution C, vortex thoroughly (for about 15 seconds) and incubate at 55oC for 10 minutes. A homogenous mixture is essential for efficient lysis. Add 500µL of the column preparation solution to each pre-assembled GenElute MiniPrep Binding column (with a red O-ring) seated in a 2mL collection tube. Centrifuge at 12’000 x g for 1 minute. Discard the eluate. Add 200µL of ethanol (95-100%) to the lysate and mix thoroughly by vortexing for 5-10 seconds. A homogenous mixture is essential. Transfer the entire contents of the tube into the binding column. Use a wide bore pipette tip to reduce shearing of the DNA when transferring the contents into the column. Centrifuge at 6’500 x g for 1 minute. Discard the collection tube containing the eluate and place the column in a new 2mL collection tube. Add 500µL of wash solution 1 to the column and centrifuge for 1 minute at 6’500 x g. Discard the 2mL collection tube containing the eluate and place the column in a new 2mL collection tube. Add 500µL of wash solution and centrifuge at maximal speed to dry out the column. The column must be free of ethanol before eluting the DNA. Centrifuge the column for an additional 1 minute at maximal speed if residue ethanol is seen. Finally, discard the collection tube containing the eluate and place the column in a new 2mL collection tube. Pipette 200µL of elution solution directly onto the center of the column and centrifuge for 1 minute at 6’500 x g to elute the DNA. Waiting 5 minutes before centrifuging by incubating at room temperature increases elution efficiency. A second repeated 5 minute incubation at room temperature followed by centrifuging at 6’500 x g will increase the yield by 20-50%. Store DNA at 2-8oC for short-term use, or -20oC for long-term use.

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