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LumiMag® BB, Magnetic Bead DNA Isolation Kit for Blood and Buccal Swab manual

The kit is designed for rapid and highly-efficient isolation of genomic DNA from whole blood and swabs using magnetic bead technology. It is compatible with KingFisher™ Flex, MagMAX™ Express-96, KingFisher™ Duo Prime, KingFisher™ mL automated platforms and can be used for manual DNA extraction using a magnetic rack.

Extracted DNA is free from protein impurities and can be used for PCR, restriction enzyme digestion, Southern blotting, preparing samples for Sanger sequencing and NGS.

套件組成

套件組成部分 數量
11753
10 minipreps 		31753
100 minipreps
B1315, Magnetic Beads (100 mg/mL), 200 uL 1 —
22850, Proteinase K (lyophilized), 10 mg 1 —
F4150, Lysis Solution BB, 4 mL 2 —
G6450, Wash Solution MAG A (with GuHCl), 5 mL 1 —
D1315, Magnetic Beads (100 mg/mL), 2 mL — 1
62850, Proteinase K (lyophilized), 100 mg — 1
P4150, Lysis Solution BB, 35 mL — 2
S6450, Wash Solution MAG A (with GuHCl), 50 mL — 1
C3850, Proteinase K Dilution Buffer, 1 mL 1 1
K2250, Wash Solution B (Concentrate to be diluted 5x with ethanol 96%), 10.0 mL 1 1
K1350, Elution Buffer (10 mM Tris-HCl, pH 8.5), 10 mL 1 1

保存期限 12 個月。

Required equipment and materials not included in the kit:

  • Automated workstation (KingFisher™ Flex, MagMAX™ Express-96, KingFisher™ Duo Prime, KingFisher™ mL)/magnetic rack
  • If using a magnetic rack: 1.5 mL Eppendorf™ or analogs tubes (two tubes for DNA isolation from one sample)
  • Ethanol 96%
  • Sodium phosphate buffer (PBS) for DNA isolation from epithelium swabs
  • Plate shaker (optional)
  • Expendable plastics for automated workstation:
KingFisher™ Flex Purification System или MagMAX™ Express-96 Deep Well Magnetic Particle Processor
95040460 96 Deep-Well Plate Microtiter deep-well
97002540 KingFisher 96 KF Plate 200 µL
97002534 KingFisher 96 tips comb for 96 DW-format
Adhesive foil for plate sealing
KingFisher™ Duo Prime
97003530 KingFisher Duo Combi pack for Microtiter Deepwell plate (plates, tips combs, and elution strips)
or
97003520 KingFisher Duo elution strip
97003500 Tips comb (12-channel) for 96 deep-well Microtiter plate
95040460 96 deep-well Microtiter plate
KingFisher™ mL
97002141 KingFisher mL Combi 240 (strips and tips combs)
or
97002111 KingFisher mL tips comb
97002121 KingFisher Strip mL

Before you start

  1. Dilute Wash Solution B concentrate 5-fold with ethanol 96% (add 4 volumes of ethanol 96% to the concentrate volume labeled on the bottle), label the cap with a mark that ethanol has been added.
  2. Add 1 mL of Proteinase K Dissolution Buffer in a tube with lyophilized Proteinase K, carefully mix, discard drops by centrifugation.
    ! Prepared proteinase K solution should be stored in a freezing chamber at −20 °C.
  3. If Lysate Solution BB or Wash Solution MAG A contains a precipitate, warm them up in a thermostat up to 50 °C and wait until completely dissolved.
  4. If using a plate shaker, choose optimal conditions for efficient mixing:
    • Make sure that the plate is tightly fixed in the shaker.
    • Add 1 mL of water to plate wells and cover the plate with foil. Determine the maximal shaker rate with which water from the wells does not splash out from the well to the foil.

Preparation of samples

Epithelium swabs:

  1. Add 1 mL of PBS in a clean 1.5 mL tube.
  2. Carefully wash the stick with the epithelium swab with PBS. Cut off the upper part of the stick and close the tube.
  3. Shake the contents of the tube using vortex for 2–3 min.
  4. Use 200 µL of the supernatant as a sample.

Whole blood: Carefully mix blood in the tube and use 200 µL as a sample.

DNA isolation with automated workstation KingFisher™ Flex, MagMAX™ Express-96

  1. Prepare plates for automated sample treatment using the instrument according to the table.
    ! To prevent contamination and solution evaporation, seal prepared plates with adhesive foil with the solutions before loading to the instrument.
Plate ID Plate position in the instrument Plate type Reagent Volume per well
Wash Plate 1 2 Deep-well Wash solution MAG A 500 µL
Wash Plate 2 3 Deep-well Wash solution B 500 µL
Elution 4 Standard Elution buffer 90 µL
Tip Comb 5 Standard Place the tips comb in the plate
  1. Carefully resuspend the magnetic bead suspension using a vortex.
  2. In an individual tube, prepare a mixture of magnetic particles with Proteinase K solution (30 µL of mixture per 1 sample = 20 µL of magnetic particle suspension + 10 µL of Proteinase K solution).
  3. Carefully mix the mixture of magnetic particles and proteinase K. Apply 30 µL aliquots of the mixture in deep plate wells.
  4. Add 200 µL of the whole blood/epithelium swab sample (see section Sample preparation) to the mixture of magnetic particles and proteinase K.
  5. Carefully mix the contents of plate wells:
    • Using the plate shaker for 2 min at the maximal rate (determined in advance, see Section Before you start),
    • or
    • Pipette, then incubate for 2 min at room temperature.
  6. Add 700 µL aliquots of Lysate Solution BB in wells.
  7. Proceed to automated sample treatment using the instrument. Choose a relevant program and start it.
    Use MagMAX_CORE program:
Instrument name Program with heating Program without heating (optional)
KingFisher™ Flex MagMAX_CORE_Flex.bdz MagMAX_CORE_Flex_no_heat.bdz
KingFisher™ 96 MagMAX™ Express-96 MagMAX_CORE_KF-96.bdz MagMAX_CORE_KF-96_no_heat.bdz
  1. According to instrument instructions, load prepared plates with the solutions (item 1) and samples (item 7) to the instrument.

Isolated DNA storage: In a freezing chamber (−20 °C), short-term storage at 4 °C.

DNA isolation using automated workstation KingFisher™ Duo Prime, KingFisher™ mL

  1. Carefully resuspend the magnetic bead suspension using a vortex.
  2. In an individual tube, prepare a mixture of magnetic particles with Proteinase K solution (30 µL of mixture per 1 sample = 20 µL of magnetic particle suspension + 10 µL of Proteinase K solution).
  3. Carefully mix the mixture of magnetic particles and proteinase K. Apply 30 µL aliquots of the mixture in plate/strip wells.
  4. Add 200 µL of the whole blood/epithelium swab sample (see section Sample preparation) to the mixture of magnetic particles and proteinase K.
  5. Carefully mix the contents of plate/strip wells:
    • Using the plate shaker for 2 min at the maximal rate (determined in advance, see Section Before you start),
    • or
    • If using KingFisher™ mL: pipette, then incubate for 2 min at room temperature.
  6. Add 700 µL aliquots of Lysate Solution BB in wells.
  7. Apply Wash Solution MAG A, Wash Solution B, Elution Buffer, and samples to corresponding plate/strip wells according to the table below.
    Load tips combs and plates/strips prepared according to the table at the same time.
KingFisher™ Duo Prime
Row ID Row in plate Plate type Reagent Volume per well
Sample A Deep-well Sample (prepared in item 6) 930 µL
Wash 1 B Wash solution MAG A 500 µL
Wash 2 C Wash solution B 500 µL
Elution Individual strip Elution strip Elution buffer 90 µL
Tip Comb H Deep-well Place the tips comb in the plate
  
KingFisher™ mL
Position ID Strip position in the instrument Well Reagent Volume per well
Sample 1 Standard Sample (prepared in item 6) 930 µL
Wash 1 2 Wash solution MAG A 500 µL
Wash 2 3 Wash solution B 500 µL
Elution 4 Elution buffer 90 µL
Tip Comb ——Place the comb in the holder.
  1. Proceed to automated sample treatment using the instrument. Choose the relevant program and start it.

Use MagMAX_CORE program:

Instrument name Program with heating Program without heating
KingFisher™ Duo Prime MagMAX_CORE_DUO.bdz MagMAX_CORE_DUO_no_heat.bdz
KingFisher™ mL — MagMAX_CORE_mL_no_heat.bdz

Isolated DNA storage: In a freezing chamber (−20 °C), short-term storage at 4 °C.

DNA isolation using a magnetic rack

  1. Carefully resuspend the magnetic bead suspension using a vortex.
  2. In an individual tube, prepare a mixture of magnetic particles with Proteinase K solution (30 µL of mixture per 1 sample = 20 µL of magnetic particles + 10 µL of Proteinase K solution).
  3. Carefully mix the mixture of magnetic particles and proteinase K. Introduce 30 µL of the mixture in individual tubes compatible with the magnetic rack.
  4. Add 200 µL of the whole blood/epithelium swab sample (see section Sample preparation) in the tube with the mixture of magnetic particles and proteinase K.
  5. Carefully mix the tube contents using a vortex. Incubate for 2 min at room temperature.
  6. Add 700 µL aliquots of Lysate Solution BB in each tube. Carefully mix using a vortex.
  7. Place the tubes in the magnetic rack. Wait until magnetic particles completely come into the sediment. Carefully discard the supernatant (for example, with a pipette).
  8. Add 500 µL of Wash Solution MAG A, carefully mix using vortex or by pipetting. Place the tubes in the magnetic rack, remove the supernatant after beads sedimentation.
  9. Add 500 µL of Wash Solution B, carefully mix using vortex or by pipetting. Place the tubes in the magnetic rack, remove the supernatant after beads sedimentation.
  10. Add 90 µL of Elution buffer, carefully mix using vortex or by pipetting. Incubate for 5 min at room temperature. Place the tubes in the magnetic rack. The supernatant contains isolated genomic DNA. Transfer the supernatant to a new clean tube.

Isolated DNA storage: In a freezing chamber (−20 °C), short-term storage at 4 °C.

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