5. Load cleared lysate
Transfer the cleared lysate from step 4 to the column prepared in step 5 and centrifuge at ≥12000 × g for 30 seconds to 1 minute. Discard the flow-through liquid.
6. Optional wash (use only with EndA+ strains)
Add 500 µl of the Optional Wash Solution to the column. Centrifuge at ≥12000 × g for 30 seconds to 1 minute. Discard the flow-through liquid.
Note: When working with bacterial strains containing the wild-type EndA+ gene, such as HB101, JM101, and the NM and PR series, the Optional Wash step is necessary to avoid nuclease contamination of the final plasmid DNA product.
7. Wash column
Important Reminder: Verify that ethanol has been added to the bottle of Wash Solution 2.
Add 750 µl of the diluted Wash Solution to the column. Centrifuge at ≥12000 × g for 30 seconds to 1 minute. The column wash step removes residual salt and other contaminants introduced during the column load. Discard the flow-through liquid and centrifuge again at maximum speed for 1 to 2 minutes without any additional Wash Solution to remove excess ethanol.
8. Elute DNA
Transfer the column to a fresh collection tube. Add 100 µl of Elution Solution or molecular biology reagent water to the column. For DNA sequencing and other enzymatic applications, use water or 5 mM Tris-HCl, pH 8.0, as an eluant. Centrifuge at ≥12000 × g for 1 minute. The DNA is now present in the eluate and is ready for immediate use or storage at -20℃.
Note: If a more concentrated plasmid DNA preparation is required, the elution volume may be reduced to minimum of 50 µl. However, this may result in a reduction in the total plasmid DNA yield.
Results
Recovery and purity may be determined by spectrophotometric analysis. The ratio of absorbance at 260 nm to 280 nm (A260/280) should be 1.7-1.9. The size and quality of DNA may be determined by agarose gel electrophoresis or pulsed field electrophoresis.
