Cat. No. D513101 / D513102 / D513103
Manual magnetic-bead workflow for bacterial culture DNA purification.
Transfer 0.5–1.5 ml bacterial culture into a 1.5 ml microcentrifuge tube. Centrifuge at 13,000 × g for 1 min to collect bacteria and remove the supernatant.
This kit is written for bacterial cultures. Pellet collection is the sample-entry step before enzymatic lysis.
Add 200 μl Buffer TE and 20 μl Lysozyme to the pellet. Mix well and incubate at room temperature for 10 min.
For Staphylococcus, add 1 μl lysostaphin (20 mg/ml) as specified in the manual.
Add 20 μl Buffer SDS, mix well and incubate at 85°C for 15 min to lyse cells.
This heat/SDS step supports bacterial cell disruption before magnetic bead binding.
If RNA-free genomic DNA is required, add 10 μl RNase A (25 mg/ml, not provided) and incubate at room temperature for 10 min.
Optional RNase treatment is displayed in the workflow but is not included in the standard cumulative timeline.
Add 500 μl Buffer MLA, 20 μl MagPure Particles and 20 μl Proteinase K. Mix by shaking at 700–900 rpm for 8 min. Place on the magnetic stand for 1 min until beads form a tight pellet, then remove the supernatant.
Proteinase K is added during the binding/lysis mixture step, together with MLA and MagPure Particles.
Add 500 μl Buffer GW1, vortex for 15 s to resuspend beads, place on the magnetic stand for 1 min and remove the supernatant.
Confirm that ethanol has been added to Buffer GW1 before use.
Add 500 μl 75% ethanol, vortex for 15 s to resuspend beads, place on the magnetic stand for 1 min and remove the supernatant.
Ethanol washing removes residual salts and wash-buffer components.
Repeat the 75% ethanol wash with 500 μl ethanol, vortex for 15 s, magnetically separate for 1 min and remove the supernatant.
Repeated ethanol washing improves removal of residual salts before drying.
Centrifuge briefly to collect liquid on the tube wall. Place the tube on the magnetic stand, carefully remove all remaining liquid and air dry for 10 min.
Avoid residual ethanol carryover, but do not over-dry the bead pellet.
Add 50–100 μl Buffer TE, resuspend the beads by vortexing and incubate at 55°C for 10 min with shaking. If no shaking device is available, vortex 2–3 times during incubation.
Elution efficiency depends on complete bead resuspension during the 55°C incubation.
Place the tube on the magnetic stand for 2 min. Transfer the supernatant containing purified DNA to a clean 1.5 ml centrifuge tube.
Avoid disturbing the bead pellet when recovering the purified DNA.
The standard cumulative timeline is approximately 74 minutes for the manual tube protocol. Optional RNase treatment, slower handling of magnetic beads, incomplete bead resuspension or additional recovery care may extend the total time. This workflow reflects the manual magnetic-bead route only; automated processing can be followed according to the official KingFisher Flex protocol in the product manual.
This workflow separates bacterial pellet collection and cell lysis from the magnetic-bead binding, washing, drying and elution path. It is intended as a practical companion to the product manual rather than a replacement for the official protocol. This note reflects the manual magnetic-bead workflow only; the automated KingFisher Flex setup is available in the official protocol and is not expanded here.
Protocol times stated in the product manual are retained where applicable. Steps without explicit timing are estimated for an experienced operator, including pipetting, tube transfer, magnetic-rack handling, supernatant removal and bead resuspension. For short protocol ranges, the timeline uses the midpoint. For long or optional protocol ranges, the displayed standard timeline uses the shortest reasonable path, while the note and total-time range indicate where extended handling may apply. Cumulative time runs continuously from bacterial collection to final DNA recovery.
D5131 uses a magnetic-particle workflow. Bacterial cells are first collected and lysed with Lysozyme and SDS heat treatment. Buffer MLA, MagPure Particles and Proteinase K then establish binding conditions so DNA adsorbs to magnetic particles. The bead complex is washed with GW1 and 75% ethanol before drying and TE elution.
The key handling points are complete pellet resuspension, effective bead resuspension during binding and wash steps, careful supernatant removal on the magnetic stand and controlled drying before elution. For Staphylococcus, lysostaphin should be included during the lysozyme step as specified in the manual. Residual ethanol may affect downstream reactions, while over-drying may make bead resuspension more difficult.