Mar 06, 2024
Version 1
Protocol: Purification of DNA from Whole Blood using the QIAamp Blood Midi Kit (Spin Protocol) + QC with Qubit fluorometer V.1
- Paula Saffie1,2,3,
- Breeana Baker4,
- Kimberley J Billingsley5,6
- 1Programa de Pos Graduacao em Ciencias Medicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil;
- 2Centro de Trastornos del Movimiento (CETRAM), Santiago, Chile;
- 3Clinica Santa Maria, Santiago, Chile;
- 4Center for Alzheimer's and Related Dementias (CARD);
- 5Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA;
- 6Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA
- Paula Saffie: Visiting fellow at Center for Alzheimer's and Related Dementias (CARD);
Protocol Citation: Paula Saffie, Breeana Baker, Kimberley J Billingsley 2024. Protocol: Purification of DNA from Whole Blood using the QIAamp Blood Midi Kit (Spin Protocol) + QC with Qubit fluorometer. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldmx3ol5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 23, 2024
Last Modified: March 06, 2024
Protocol Integer ID: 93935
Keywords: QIAGEN, Midi KIt, DNA extraction
Funders Acknowledgement:
Center for Alzheimer's and Related Dementias (CARD)
Grant ID: not applicable
Disclaimer
‱ QIAGEN 2013–24.
Thermo Fisher Scientific Inc
Abstract
The QIAamp DNA Blood Midi procedure yield pure DNA ready for direct restriction digestion or amplification. With a suitable centrifuge rotor, eight samples can be prepared simultaneously in about 1.5 hours with approximately 30 minutes of hands-on time.
QIAamp DNA Blood Midi Kits are suitable for use with whole blood that has been treated with EDTA, citrate, or heparin, and samples may be fresh or frozen. Separation of leukocytes is not necessary. The procedure require no phenol/chloroform extraction or alcohol precipitation and involve
minimal handling.
DNA is eluted in Buffer AE or water and is ready for direct addition to PCR or other enzymatic reactions, or it can be safely stored at –30 to –15°C for later use.
The purified DNA is virtually free of protein, nucleases, and other contaminants or inhibitors of
downstream applications.
DNA ranges in size up to 50 kb, with fragments of approximately 30 kb predominating.
Source: QIAamp DNA Blood Midi/Maxi Handbook
Attachments
Materials
Equipment and Reagents to Be Supplied by User
- Suitable lab coat, disposable gloves
- Pipets + tips for each: P5000, P1000, P200, P10
- Centrifuge capable of attaining 4500 x g (5000 rpm), equipped with a swing-out rotor and buckets that can accommodate 15 ml centrifuge tubes
- 15 ml Falcon tubes
- Racks
- Water bath, heated to 70°C
- Vortex
- Mini centrifuge
- Stiled water
- Ethanol (96–100%): Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone
- Paper towels for cleaning
- Spray bottles with ethanol for cleaning
- Biohazard waste bags
- Airflow Benchtop Hood
- Fridge
- Freezer
- AC for the lab
- Access to Ice
- Buckets for Ice
- Qubit‱ Fluorometer (4 or flex)
- Qubit‱ 1X dsDNA High Broad Range (BR) Assay Kits: Working solution + Assay standards
- Qubit Assay Tubes
Qubit 4 (1 sample) and Qubit flex (8 samples)
Kit Contents
QIAamp Midi Spin Columns
Collection Tubes (15 ml)
Buffer AL*
Buffer AW1* (concentrate)
Buffer AW2 (concentrate)
Buffer AE
QIAGEN‱ Protease: Resuspension volume 4.4 ml or 5.5 ml depending on the kit units (20 or 100)
* Not compatible with disinfecting agents containing bleach. Contains a chaotropic salt. Take appropriate safety
measures, and see page 5 for further safety information.
† Resuspension volume 4.4 ml
‡ Resuspension volume 5.5 ml
Preparation of reagents
- QIAGEN Protease (store at 2–8°C stable for 2 months, storage –30 to –15°C will prolong the life)
Pipet 4.4 ml distilled water into the vial of lyophilized QIAGEN Protease provided in the QIAamp DNA Blood Midi Kit (20), or 5.5 ml in the QIAamp DNA Blood Midi Kit (100), as indicated on the labels.
- Buffer AL* (store at room temperature 15–25°C, estable 1 year)
Mix Buffer AL thoroughly by shaking before use.
- Buffer AW1 (store at room temperature, 15–25°C, estable 1 year)
Supplied as a concentrate. Add the appropriate amount of ethanol (96–100%) as indicated on the bottle (only
first time)
- Buffer AW2 (store at room temperature, 15–25°C, estable 1 year)
Supplied as a concentrate. Add the appropriate amount of ethanol (96–100%) as indicated on the bottle (only
first time)
Before start
■ Equilibrate samples to room temperature (15–25°C) before starting.
■ Prepare a 70°C water bath (critical initial step and must precede all others in the protocol, Failure to execute prior to proceeding may compromise the integrity and validity of the entire procedure).
■ Ensure that Buffer AW1, Buffer AW2, and QIAGEN Protease have been prepared (Detailed on "materials").
■ If a precipitate has formed in Buffer AL, redissolve by incubating at 56°C.
DNA extraction procedure
DNA extraction procedure
Summarized procedure
Pipet200 µL QIAGEN Protease into the bottom of a 15 ml centrifuge tube. Add 2 mL Sample and mix briefly.
Add2.4 mL Buffer AL, and mix thoroughly by inverting the tube 15 times, followed by additional vigorous shaking for at least 1 min (Check that the tubes are tightly closed).
Incubate at 70 °C 10 min.
(During this time, store Protease in the fridge)
Carefully transfer (just squirt solution in middle of the membrane, don't need to go inside the tube with the tip) one half (aprox2-3 mL ) of the solution onto the QIAamp Midi column placed in a 15 ml centrifuge tube, cerefully not to moistening the rim.
With the cap tightly closed, centrifuge 3000 rpm (1850 x g) for 3min.
Add2 mL ethanol (96–100%) to the sample, and mix by inverting the tube 10 times, followed by additional vigorous shaking.
Remove the QIAamp Midi column, discard the filtrate (dab the rim to remove any exccess of liquid), and place the QIAamp Midi column back into the 15 ml centrifuge tube. Load the remainder of the solution onto the QIAamp Midi column.
With the cap tightly closed, centrifuge 3000 rpm (1850 x g) for 3min.
Remove the QIAamp Midi column, discard the filtrate, and place the QIAamp Midi column back into the 15 ml centrifuge tube.
Add 2 mL Buffer AW1 (carefully, without moistening the rim) to the QIAamp Midi column.
With the cap tightly closed, centrifuge at 5000 rpm ( 4500 x g) for 1 min.
Add 2 mL Buffer AW2 (carefully, without moistening the rim) to the QIAamp Midi column.
With the cap tightly closed, centrifuge at 5000 rpm (4500 x g) for 15 min.
Place the QIAamp Midi column in a clean 15 ml centrifuge tube, and discard the collection tube containing the filtrate.
Pipet300 µL Buffer AE directly onto the membrane of the QIAamp Midi column and close the cap.
Centrifuge at 5000 rpm (4500 x g) for 2 min.
Pipet 300 µL Buffer AE directly onto the membrane of the QIAamp Midi column and close the cap.
Incubate at room temperature for 5 min.
Centrifuge at 5000 rpm (4500 x g) for 2 min.
Sample QC procedure (Qubit Fluorometer)
Sample QC procedure (Qubit Fluorometer)
STANDARDIZE THE QUBIT (every time you use it or once a week according to lab protocol)
Get0.2 mL Qubit tubes.
In one strip, add10 mL of BR Standard 1.
In the other strip, add10 mL of BR Standard 2.
Add 190 µL of the working solution to all of the tubes.
Vortex, spin down, and incubate in the dark for about 2 min to optimize the reaction.
On the Home screen, press to select the Assay to perform (dsDNA)
Press Read standards & run samples to read new standards
When prompted, load the Qubit‱ Flex Tube Strip containing Standard #1 into
the sample chamber, then press Run standards. The reading takes ~3 seconds.
When prompted, load the Qubit‱ Flex Tube Strip containing Standard #2 into
the sample chamber, then press Run standards. The reading takes ~3 seconds.
If your calibration is successful, press Next to proceed to “Read samples” or begin in this step if it is already calibrated.
READ SAMPLES
Add198 µL or 199 µL of dsDNA Broad Range Working Solution to a 0.2 mL Qubit tube.
Add2 µL or1 µL of Sample thoroughly mixed (flick + spin down) in the Qubit tube (200 µL total amount )
Pulse-vortex and spin down. Incubate in dark for at least 2min.
Load the tube strip containing the samples as shown. If you have fewer than 8 samples, press to deselect the tube positions that do not contain a sample.
Press Output sample units to open the Output Units screen, then select the desired units. Press Next to go to the Sample volume screen.
Select the units accordingly
In the Sample volume screen enter the sample volume used: it could be 1 µL or 2 µL .
Press Run samples. The reading takes approximately 3 seconds.
Expected result
To display the results in list view, press the Graph button to hide the graph.
The Results screen shows the concentration of each original sample in a list
form, using the output units selected at the beginning of the assay.
On average, the Midi kit yields about 20-30 micrograms of DNA per sample. In this example, you can find this quantity by multiplying the Qubit concentration (104 nanograms per microliter) by the sample volume (550 microliters), then dividing the result by 1000. In this case, it equals 57.20
| Post-Ext. Qubit(ng/ul) | Post-Ext. Volume(ul) | Post-Ext. Amount (ug) | |
| 104 | 550 | 57.20 |
Protocol references