Nov 21, 2022
Manual QIAgen DNA Extraction
- Clemens Scherzer1,2,
- Bradley Hyman3,2,
- Charles Jennings1,2
- 1Brigham and Women's Hospital;
- 2Harvard Medical School;
- 3Massachusetts General Hospital
Protocol Citation: Clemens Scherzer, Bradley Hyman, Charles Jennings 2022. Manual QIAgen DNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw5z12vmk/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: February 18, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 47414
Keywords: qiagen, DNA, extraction, ASAPCRN
Abstract
This protocol explains the Standard Operating Protocol for manually extracting DNA using Qiagen.
Guidelines
FREEZER STORAGE
Freezers are divided into 4 shelves, with 6 racks per shelf, and 24 boxes that can be held in each shelf. In total, 576 boxes, approximately 2,160 sample sets, can be stored in one -80°C freezer. The first three shelves are designated by visit number: Shelves A1-6 (top shelf) house samples from enrollment visits, shelves B1-6 (2nd shelf) house samples from the 1st year follow-up, and shelves C1-6 (3rd shelf) house samples from the 2nd year follow-up. Shelves D1-6 contain packed red blood cell tubes (PRBC), DNA, and RNA, extracted from blood as described in the protocols above. CSF is designated between two freezers in selected racks. Freezer storage and transactions of samples are recorded in the Freezerworks Inventory software.
Materials
MATERIALS:
- Buffy Coat tube (from WHOLE BLOOD, PLASMA, BUFFY COAT PROCESSING)
- 1.5mL low-retention microcentrifuge tubes (Fisher Scientific, Cat #02-681-320)
- QIAamp DNA Blood Mini Kit (250) (QIAgen)
- Freezerbonds Labels (Fisher Scientific, Cat #22500521)
Safety warnings
Please refer to Safety Data Sheets (SDS) for health and environmental hazards. Gain all required consent and experimental approvals before beginning any procedures.
Before start
DNA Q/C GOALS
1. Cary Concentration Assay
a. 260/280 = 1.8-2.0
b. Manual Puragene Extraction: 260 µg /mL (65 µg total) of DNA/subject
c. Automated QIAcube Extraction: 125 µg/mL (50 µg total) of DNA/subject
2. .7% Agarose Gel Electrophoresis
a. Human DNA = 23.13 kb with λ DNA-HindIII digest (NEB)
Manual Qiagen DNA Extraction
Manual Qiagen DNA Extraction
20m 30s
20m 30s
Thaw buffy coats or whole bloods by equilibrating to Room temperature .
Heat a heating block to 56 °C .
Gather all the necessary reagents/buffers (Buffer AE, Buffer AW1, Buffer AW2, Buffer AL, 100% ethanol, Qiagen Protease).
If a precipitate has formed in Buffer AL, dissolve by incubating in 56 °C .
Gather appropriate number of QIAamp Mini spin columns and 2ml collection tubes.
Pipet 20 µL Qiagen protease into the bottom of a 1.5ml microcentrifuge tube.
Add 200 µL BC/WB to the 1.5ml microcentrifuge tube.
Add 200 µL Buffer AL to the sample and mix by pulse-vortexing for 00:00:15 at half-speed.
Note
To ensure efficient lysis, it is essential that the sample and Buffer AL are mixed thoroughly to yield a homogeneous solution. Add each sample and then AL immediately after.
15s
Incubate on 56 °C heat block for 00:10:00 .
Note
DNA yield reaches a maximum after lysis for 10 minutes at 56C. Longer incubation times have no effect on yield or quality of the purified DNA.
10m
Briefly centrifuge the 1.5ml microcentrifuge tubes to remove drops from the inside of the lid.
Add 200 µL 100% ethanol to the sample, and mix again by pulse-vortexing for 00:00:15 . After mixing, briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid.
15s
Carefully add the sample from step 6 to the QIAamp Mini spin column (in a 2ml collection tube) without wetting the rim. Close the cap, and centrifuge at 20000 x g, 00:01:00 , (14,000rpm) .
Place in a clean 2 mL collection tube.
Carefully open the QIAamp Mini spin column and add 500 µL Buffer AW1 without wetting the rim. Close the cap and centrifuge at 6000 x g, 00:01:00 . Place the QIAamp Mini spin column in a clean 2ml collection tube and discard the collection tube containing the filtrate.
Carefully open the QIAamp Mini spin column and add 500 µL Buffer AW2 without wetting the rim. Close the cap and centrifuge at 20000 x g, 00:03:00 , (14,000rpm) .
Place the QIAamp Mini spin column in a new 2ml collection tube and discard the old collection tube with the filtrate. Centrifuge at 20000 x g, 00:01:00 , (14,000rpm) .
Note
This is a dry spin.
Place the QIAamp Mini spin column in a clean 1.5ml microcentrifuge tube labeled with HBS IDs and discard the collection tube containing the filtrate. Carefully open the QIAamp Mini spin column and add 150 µL Buffer AE . Incubate at Room temperature for 00:05:00 and then centrifuge at 6000 x g, 00:01:00 , (8,000rpm) .
5m
Repeat step 11 for a second elution of 150μl of DNA.
Combine the two 150μl aliquots and mix and split them.