Transfer the supernatant to a fresh microcentrifuge tube and add 10 µl 10% SDS (prepared from 20% SDS supplied with the kit) to the lysate.

For tissues, cut the tissue into smaller pieces and ensure the tissue is completely immersed in the Lysis Buffer to obtain optimal lysis. Samples on the gel are:   Lane 1: 36.7 ng of DNA isolated from E. coli (5 x 108 cells) Lane 2: Blank Lane 3: 21.8 ng DNA isolated from human HeLa (1x106 cells) Lane 4: 19.3 ng DNA isolated from human Huh-7 (2x106 cells) Lane 5: 37.5 ng DNA isolated from human 293F (1x106 cells) Lane 6: 60.3 ng DNA isolated from mouse liver (25 mg) Lane 7: 69.1 ng DNA isolated from mouse tail (0.5-cm section) Lane 8: Blank Lane 9: 1 Kb DNA Extension Ladder (0.5 µg/lane). Place the sample on the Magnetic Separator for 30 seconds to 1 minute. Calculate the concentration of DNA using the formula: Decrease the amount of starting material used. Repeat Steps 1-2, once. To aspirate the supernatant after bead washing, place the pipette tip away from the beads by angling the pipette such that the tip is pointed away from the pellet and carefully remove the supernatant without disturbing or removing any beads. The yield is the total yield from 2 x 200 µl elution.

Store beads at room temperature. The manual also contains protocols for reaction cleanup and extraction of gDNA from … The PureLink™ Genomic DNA Purification Kit allows rapid and efficient purification of genomic DNA. Add Protease Buffer and Protease to the tube and vortex the capped tube to thoroughly disperse the bead pellet (~30 seconds). Without removing the plate from the Magnetic Separator, carefully remove and discard the supernatant using a 1 ml pipette without disturbing the pellet of beads by angling the pipette such that the tip is pointed away from the pellet. Add 20 µl Proteinase K solution (supplied with the kit) to lyse the cells. Add 20 µl Proteinase K (supplied with the kit) to the sample and mix well by vortexing. Blood collected in the presence of anti-coagulants such as EDTA, heparin, or citrate. Keep the tube on the Magnetic Separator for an additional 1 minute to allow any remaining liquid to settle to the bottom of the tube. Add 0.5 ml Protease Buffer and 10 µl Protease to the samples and gently agitate the plate by swirling or placing the plate on a shaker set to low speeds until the magnetic bead pellet is completely dispersed.

Binding DNA Follow the procedure below to bind DNA to the GeneCatcher™ Magnetic Beads. Be sure to add 96–100% ethanol to Wash Buffer (W5). Remove any particulate or viscous material by centrifugation prior to loading the lysate on to the spin cartridge.

Agitate the samples by gently swirling the plate to resuspend any settled beads. Add 200 µl Binding Buffer (L3) supplied with the kit to the lysate. Mix the beads by gently swirling the tube until the beads are evenly distributed.

For high DNA content, incubate the samples for an additional 30 minutes. Incubate at room temperature for 2 minutes.

Place the sample on the 24-well magnetic Separator for 1 minute. Do not use water to elute DNA. The supernatant should be clear, olive green color.

The procedure is designed for isolating gDNA using the GeneCatcher™ Magnetic Beads procedure. Centrifuge cartridge at room temperature at 12,000 x g for 30 seconds. Use ChargeSwitch. We recommend using the 24-well Magnetic Separator (catalog No.

Mix the sample properly with Binding Buffer and ethanol by vortexing. To remove Wash Buffer (W5), discard Wash Buffer (W5) flow through. Place the tube on a 50 ml Tube Magnetic Separator Rack (see above for a figure) for 3 minutes. Incomplete dissociation of DNA from the ChargeSwitch. Remove the tube containing the pelleted magnetic beads (Step 12, above) from the Magnetic Separator. To purify genomic DNA from 2 ml blood sample, use the specified reagent volumes for 3 ml blood samples as described in the protocol.

Add 200 µl of Elution Buffer (E1) or sterile, distilled water (pH >7.0) to the cartridge. The SDS precipitates in the presence of guanidine isothiocyanate. Not for use in diagnostic procedures. Add elution buffer and perform incubation for 1 minute with elution buffer before centrifugation. Add 250 µl Elution Buffer (E5; 10 mM Tris-HCl, pH 8.5) to the samples and gently agitate the plate by swirling or placing the plate on a shaker set to low speeds to dislodge the pellet. Contents The components supplied in the GeneCatcher™ gDNA Blood Kits are listed below. To avoid repeated freezing and thawing of DNA, store the purified DNA at 4° C for immediate use or aliquot the DNA and store at -20° C for long-term storage. Dried beads are non-functional. DNA Quality Genomic DNA isolated from various samples was analyzed by agarose gel electrophoresis on a 0.8% E-Gel® agarose gel. Remove the supernatant without disturbing the pellet of beads by angling the pipette tip away from the pellet. Other magnetic separators may not provide similar magnetic strength or may not be compatible with the volumes used in the protocol. To recover more DNA, perform a second elution step with 200 µl Elution Buffer (E1) or sterile, distilled water (pH >7.0) using another sterile 1.5 ml microcentrifuge tube. The yield and quality of DNA isolated is dependent on the type and age of the starting material. For cells, harvest cells and resuspend cell pellet in 180 µl Lysis Buffer (L6) and 20 µl Proteinase K. Incubate at 55°C until lysis is complete. Without removing the plate from the Magnetic Separator, carefully remove and discard the supernatant using a 1 ml pipette without disturbing the pellet of beads. Optional: Add 20 µl RNase A (supplied in the kit) to lysate and incubate at room temperature for 2 minutes. Do not freeze the beads as they will become irreparably damaged. Maintain a sterile environment while working to avoid any contamination from DNases. Materials Needed.

Add 2.5 ml Lysis Buffer (L13) to the wells and mix the beads by gentle agitation of the plate using a plate shaker set to low speeds (800-1,000 rpm) or by gentle swirling. Add 10 µl 10% SDS (prepared from 20% SDS supplied with the kit) to the lysate and mix immediately by vortexing for 5 seconds to denature proteins.

The extraction kit is designed for pelleted cells from either cultured eukaryotic cell lines or purified peripheral blood mononuclear cells (PBMC). Increase the length of incubation at room temperature. You may need to optimize lysis conditions prior to DNA purification to obtain the best results for your specific sample. Place the sample on the 24-well magnetic Separator for 3 minutes. Centrifuge the lysate at maximum speed for 5 minutes at room temperature to remove any particulate materials. Introduction The DNA yield obtained from various samples is described below. Centrifuge the cartridge at maximum speed for 1.5 minute at room temperature.

Note: If the supernatant is discolored, repeat Steps 6-7.