Dinh/Dinh 2015/NOTES/2015-9-14

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Plasma DNA purification using QIAamp Circulating Nucleic Acid Kit[edit]

  • Received 2 aliquots from Alan.
  • Things to do before starting:
Equilibrate samples to RT
Prepare fresh 1X PBS to bring all samples to the same volumes
Set up the QIAvac 24.
Set a water bath to 60C for use with 5 mL tubes.
Set up a heating block to 56C for use with 2 mL collection tubes.
Equilibrate Buffer EB to room temperature for elution (manual says to use Buffer AVE, but Noi uses Buffer EB).
Ensure that Buffer ACB, Buffer ACW1, and Buffer ACW2 have been prepared according to instructions.
Add 40 mL isopropanol (100%) to 60 mL buffer ACB. Mix well after adding isopropanol in 2x50 mL tubes.
Add 25 mL ethanol (100%) to 19 mL buffer ACW1. Mix well after adding ethanol.
Add 30 mL ethanol (100%) to 13 mL buffer ACW2. Mix well after adding ethanol.
Add carrier RNA reconstituted in Buffer AVE to Buffer ACL according to instructions:
40 mL ACL + 250 uL carrirer RNA (dissolved in AVE, 0.2 ug/uL).
  • Most time consuming step is pooling the aliquots and measuring out PBS to add to make 1 mL.
Sample IDE17DA1:D32 Total plasma volume PBS volume to make 1 mL Proteinase K
UCSD-006.11 231 769 100
UCSD-006.12 670 330 100
UCSD-006.13 690 310 100
UCSD-006.14 585 415 100
UCSD-006.15 610 390 100
UCSD-006.16 650 350 100
UCSD-006.17 480 520 100
UCSD-006.18 425 575 100
UCSD-006.19 650 350 100
UCSD-006.20 641 359 100
UCSD-006.21 670 330 100
UCSD-006.22 460 540 100
UCSD-006.23 900 100 100
UCSD-006.24 725 275 100
UCSD-006.25 150 850 100
UCSD-006.26 550 450 100
UCSD-006.27 55 945 100
UCSD-006.28 940 60 100
UCSD-006.29 940 60 100
UCSD-006.30 405 595 100
UCSD-007.11 595 405 100
UCSD-007.12 535 465 100
UCSD-007.13 630 370 100
UCSD-007.14 600 400 100
UCSD-007.15 455 545 100
UCSD-007.16 630 370 100
UCSD-007.17 355 645 100
UCSD-007.18 430 570 100
UCSD-007.19 760 240 100
UCSD-007.20 550 450 100
UCSD-007.21 620 380 100
UCSD-007.22 385 615 100
UCSD-007.23 540 460 100
UCSD-007.24 685 315 100
UCSD-007.25 700 300 100
UCSD-007.26 690 310 100
UCSD-007.27 700 300 100
UCSD-007.28 780 220 100
UCSD-007.29 690 310 100
UCSD-007.30 398 602 100
  • Protocol steps:
  1. . Add 100 uL of Proteinase K into the 5 mL labeled tubes.
  2. . Add 1 mL of plasma/serum to the tube. If not enough volume, add 1X PBS to make 1 mL
  3. . Add 800 uL of Buffer ACL with carrier RNA added.
  4. . Cap and mix by pulse vortexing for 30s to yield a homogenous solution.
  5. . Incubate at 60C for 30 minutes. Set up the QiaVac in this time.
  6. . Add 1.8 mL Buffer ACB to the lysate and pulse vortex for 15-30 s.
  7. . Incubate the lysate-Buffer ACB mixture for 5 minutes on ice. Samples can be left on ice while waiting for room on the QiaVac.
  8. . Carefully apply the lysate-Buffer ACB mixture into the tube extender of the QIAamp Mini columns. Let all lysates get drawn through the columns completely.
  9. . Carefully remove and discard the tube extender.
  10. . Apply 600 uL Buffer ACW1 to the QIAamp mini column. Leave the lid off the column and Let all the buffer get drawn through. Turn off the vacuum and #. release pressure by opening one of the valves.
  11. . Apply 750 uL Buffer ACW2 to the column, leave the lid off the column and let all the buffer get drawn through as before. Turn off the vacuum and release pressure by opening one of the valves.
  12. . Apply 750 uL 100% ethanol (instead of 100% ethanol we mistakenly used 100% isopropanol instead) through the column and let all the buffer get drawn through as before. Turn off the vacuum and release pressure by opening one of the valves.
  13. . Close the lid of the columns, LABEL the columns, and remove them from the vacuum.
  14. . Place the QIAamp Mini column in a clean 2 mL collection tube, and centrifuge at full speed for 3 min.
  15. . Place the columns into new 2 mL collection tube, and incubate at 56C for 10 minute with lids open.
  16. . Place the column in a clean 1.5 mL elution tube and apply 30 uL EB twice to the center of the membranes. Close and incubate for 3 min. Total volumes after elution should be ~60 uL each.
  17. . Centrifuge for 1 min at full speed to elute. Label the tubes before discarding the columns.
  • Notes: I didn’t get all of the lysate-Buffer ACB mixture into the columns by just pouring them, so I pooled the leftover contents together and got about 2.8 mL of the mixtures. I ran the mixtures through two QIAamp mini columns and purified the DNA for use as a testing control. Due to the final wash with isopropanol instead of ethanol, my salt content was probably very high in all the samples. I nanodrop the combined plasma DNA sample and found that DNA was pulled through however.
  • Perform ethanol precipitation of the flow-through to remove salts.
  1. . Ethanol precipitation:
  2. . Add nuclease free water to each sample to make 200 uL total volume.
  3. . Add 2 uL of GlycoBlue to each tube.
  4. . Add 20 uL of 3 M sodium acetate.
  5. . Add 500 uL of 100% ethanol to each sample.
  6. . Leave in -80C freezer for 1 hour. Centrifuge for 45 minutes at 10,000 g (4C).
  7. . Wash with ice-cold 75% ethanol. Centrifuge for 15 mintues at 10,000 g (4C).
  8. . Elute with 30 uL EB buffer
  • Performed the ethanol precipitation and quantification on 6 samples + 1 test sample first to check the yield (9/15/15). The yield from this kit was actually very good. Most of the samples has greater than 10 ng DNA.
Sample ID Initial Conc. (ng/uL) ng yielded Post ETOH PPT, Conc. (ng/uL) ng yielded % yield after ETOH PPT
6.12 0.302 18.12 0.364 11.284 62%
6.23 1.854 111.24 0.846 26.226 24%
6.29 0.266 15.96 0.35 10.85 68%
7.11 0.366 21.96 0.36 11.16 51%
7.2 0.444 26.64 0.756 23.436 88%
7.28 1.094 65.64 1.266 39.246 60%
Combined 0.452 27.12 1.644 50.964 188%
Plasma Sample Sample Concentration (ng/uL) Volume eluted in EB (uL) Yield (ng) Plasma volume (uL) Est. yield from 1 mL (ng)
ALL 0.94 30 28.08 NA NA (probably all contributed by 7.24 sample).
6.11 0.25 30 7.38 231 31.95
6.12 0.31 30 9.36 670 13.97
6.13 1.04 30 31.26 690 45.30
6.14 0.48 30 14.28 585 24.41
6.15 0.19 30 5.82 610 9.54
6.16 0.39 30 11.76 650 18.09
6.17 0.27 30 7.98 480 16.63
6.18 0.32 30 9.48 425 22.31
6.19 0.39 30 11.82 650 18.18
6.20 0.18 30 5.40 641 8.42
6.21 0.38 30 11.40 670 17.01
6.22 1.29 30 38.64 460 84.00
6.23 0.88 30 26.34 900 29.27
6.24 0.37 30 10.98 725 15.14
6.25 0.23 30 6.96 150 46.40
6.26 0.33 30 9.84 550 17.89
6.27 0.17 30 5.10 55 92.73
6.28 4.08 30 122.40 940 130.21
6.29 0.31 30 9.42 940 10.02
6.30 0.43 30 12.84 405 31.70
7.11 0.42 30 12.66 595 21.28
7.12 0.16 30 4.68 535 8.75
7.13 0.32 30 9.66 630 15.33
7.14 0.37 30 11.10 600 18.50
7.15 0.32 30 9.66 455 21.23
7.16 0.29 30 8.82 630 14.00
7.17 1.09 30 32.58 355 91.77
7.18 0.71 40 28.24 430 65.67
7.19 0.44 30 13.20 760 17.37
7.20 0.75 30 22.38 550 40.69
7.21 0.37 30 11.04 620 17.81
7.22 0.77 30 23.04 385 59.84
7.23 1.20 40 48.00 540 88.89
7.24 106.00 40 4240.00 685 6189.78
7.25 0.29 30 8.76 700 12.51
7.26 0.26 30 7.92 690 11.48
7.27 0.55 30 16.50 700 23.57
7.28 1.06 30 31.80 780 40.77
7.29 0.28 30 8.28 690 12.00
7.30 2.32 30 69.60 398 174.87