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Higher gDNA Concentrations with the Perfect gDNA Blood Mini Kit ,,, by Varying the Final Elution Volume

l Solution G2 to the tube containing the supernatant. Vortex the sample for 5 seconds. Place a spin column in a fresh microcentrifuge tube. Transfer the sample to the spin column assembly. Incubate the sample at room temperature for 1 minute.
  • Centrifuge the sample for 2 minutes at 12,000 to 16,000 x g. Remove the spin column and decant flowthrough. Place the spin column back into the same tube.
  • Add 600 l Diluted Wash Buffer to the spin column. Centrifuge for 1 minute at 12,000 to 16,000 x g. Remove the spin column and decant flowthrough. Place the spin column back into the same tube.
  • Add 400 l Diluted Wash Buffer to the spin column. Centrifuge for 3 minutes at 12,000 to 16,000 x g. Carefully remove the spin column without splashing the Wash Buffer onto the bottom of the column. Place the spin column into a fresh microcentrifuge tube.
  • Add 50l to 200 l Elution Buffer to the spin column, making sure that the buffer comes into contact with the spin column filter. Incubate the sample at 70C for 3 minutes. Centrifuge for 1 minute at 12,000 to 16,000 x g to elute gDNA. Store purified gDNA at 4C.
    • Restriction digests
    With a decrease in elution volume, an increase in concentration of contaminants may be expected. Restriction digests were performed to test the purity of the DNA when eluting in smaller volumes. 200 ng of sample was incubated with 2 and 5 units of Sau3AI (New England Bio-labs) and without enzyme at 37C.

    PCR
    gDNA was isolated from whole blood preserved in EDTA of three individuals. DNA was eluted in 50 l, 100 l, and 200 l for each individual. 50 ng and 200 n
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