Methods for nucleic acid isolation and kits using a microfluidic device and concentration step
A microfluidic device, nucleic acid technology, which is effective in the field of separating nucleic acids from samples
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Embodiment 1
[0193] Example 1: Method for Isolating Genomic DNA from Whole Blood Without Using Chelating Solid Phase Materials
[0194] Add one (1) μL of pure TRITON X-100 to one hundred (100) μL of whole blood. The solution was incubated at room temperature (about 21° C.) for about 5 minutes, and the solution was vortexed intermittently (for about 5 seconds every 20 seconds). The solution was observed to ensure it was clear before proceeding to the next step. The solution was centrifuged at 400 rcf for approximately 10 minutes in an Eppendorf Model 5415D centrifuge. Separate and discard the supernatant, leaving approximately two (2) μL of the concentrated material at the bottom of the centrifuge tube. Transfer this concentrated material to a new microcentrifuge tube. Ten (10) μL of 0.1 M NaOH was added, mixed and incubated at room temperature (approximately 21° C.) for approximately 5 minutes. A 2 μL aliquot was removed and added to 10 μL of 40 mM TRIS-HCl, pH 7.4.
Embodiment 2A
[0195] Example 2A: Effect of Inhibitor / DNA on PCR: Varying Inhibitor Concentration at Fixed DNA Concentration
[0196] Serial dilutions of inhibitors were made before spiking pure human genomic DNA in order to study the effect of inhibitors on PCR. To 10 μL of 15 nanogram / microliter (ng / μL) human genomic DNA, add 1 μL of different Mix I (pure or its dilutions) (sample 2-no inhibitor added, 2D-pure, 2E-1 :10, 2F-1:30, 2G-1:100, 2H-1:300) and vortex. Two (2) μL aliquots of each sample were taken for 20 μL PCR. The results are shown in Table 2.
[0197] Mix I: Add 1 μL of pure TRITON X-100 to one hundred (100) μL of whole blood. The solution was incubated at room temperature (about 21° C.) for about 5 minutes, and the solution was vortexed intermittently (for about 5 seconds every 20 seconds). The solution was observed to ensure it was clear before proceeding to the next step. The solution was centrifuged at 400 rcf for approximately 10 minutes in an Eppendorf Model 5415D ce...
Embodiment 2B
[0198] Example 2B: Effect of Inhibitor / DNA on PCR: Varying DNA Concentration at Fixed Inhibitor Concentration
[0199] To 10 μL of human genomic DNA was added 1 μL of a 1:3 dilution of Mix I (as described above). The DNA concentrations studied were as follows: samples 2J-15 ng / μL, 2K-7.5 ng / μL, 2L-3.75 ng / μL, 2M-1.5 ng / μL. Two (2) μL aliquots were taken from each sample for 20 μL PCR. The results are shown in Table 2.
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