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:
- . Add 100 uL of Proteinase K into the 5 mL labeled tubes.
- . Add 1 mL of plasma/serum to the tube. If not enough volume, add 1X PBS to make 1 mL
- . Add 800 uL of Buffer ACL with carrier RNA added.
- . Cap and mix by pulse vortexing for 30s to yield a homogenous solution.
- . Incubate at 60C for 30 minutes. Set up the QiaVac in this time.
- . Add 1.8 mL Buffer ACB to the lysate and pulse vortex for 15-30 s.
- . Incubate the lysate-Buffer ACB mixture for 5 minutes on ice. Samples can be left on ice while waiting for room on the QiaVac.
- . 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.
- . Carefully remove and discard the tube extender.
- . 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.
- . 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.
- . 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.
- . Close the lid of the columns, LABEL the columns, and remove them from the vacuum.
- . Place the QIAamp Mini column in a clean 2 mL collection tube, and centrifuge at full speed for 3 min.
- . Place the columns into new 2 mL collection tube, and incubate at 56C for 10 minute with lids open.
- . 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.
- . 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.
- . Ethanol precipitation:
- . Add nuclease free water to each sample to make 200 uL total volume.
- . Add 2 uL of GlycoBlue to each tube.
- . Add 20 uL of 3 M sodium acetate.
- . Add 500 uL of 100% ethanol to each sample.
- . Leave in -80C freezer for 1 hour. Centrifuge for 45 minutes at 10,000 g (4C).
- . Wash with ice-cold 75% ethanol. Centrifuge for 15 mintues at 10,000 g (4C).
- . 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 |