JHU Oncology Microarray Facility
DNA Preparation Protocol and Submission
Core is able to isolate DNA from different types of samples for our customers. Consult core
before you isolate your samples.
Core recommends using DNeasy Blood & Tissue Kit (Qiagen, cat. #69504) for DNA isolation. The
following procedures are adopted from kit’s handbook. Consult handbook for additional details.
Tissue samples
1. Place 10-25 mg frozen tissue piece into 1.5 ml Eppendorf tube (or cut into smaller pieces under
frozen condition if desired prior to this step).
2. Add 180 l Buffer ATL and 20 l proteinase K provided in the kit.
3. Mix thoroughly by gentle vortex or inversion.
4. Incubate at 56oC until the tissue is completely lysed (incubation can take 3 hours to overnight
depend on tissue type).
5. Gently vortex occasionally during incubation.
6. Add 4 l RNase A (100 mg/ml), mix by gentle vortex or inversion and incubate for 5 min at
room temperature.
7. Centrifuge at 6000xg for 1 min to spin down insoluble debris and transfer solution to a new
tube.
8. Add 200 l Buffer AL to the sample and mix thoroughly by gentle-vortex or inversion.
9. Add 200 l of 100% ethanol and mix thoroughly by gentle-vortex or inversion.
10. Pipet the mixture (including any precipitate) into the DNeasy Mini spin column placed in a 2
ml collection tube. Centrifuge at 6000xg for 1 min. Discard flow-through.
11. Add 500 l Buffer AW1, and centrifuge for 1 min at 6000xg. Discard flow-through.
12. Add 500 l Buffer AW2, and centrifuge for 3 min at maximum speed. Discard flow-through
and collection tube. To assure drying the column, a second brief centrifuge or leave the column
with tube-lid open for 5 min may be preferred.
13. Place the DNeasy Mini spin column in a microcentrifuge tube, pipet 30 - 50 l Buffer AE into
column. Incubate at room temperature for 1 min, and then centrifuge for 1 min at 6000xg to
elute DNA.
14. To maximize DNA yield, elution can be done with adding another 50 l nuclease-free water
and concentrate the combination of 2 elutes by speed-vacuum.
Cultured or FACS-sorted cells
1. Use DNeasy Blood & Tissue Kit (Qiagen, cat. # 69504) for DNA isolation.
2. Spin down appropriate number of cells (down to 1x 104 for sorted cells or up to 2x 106 for
cultured cells) for 5 min at 300xg. Re-suspend cell pellet in 180 l 1x PBS. Add 20 l
proteinase K.
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Sample preparation and submission guideline
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February 2012
JHU Oncology Microarray Facility
3. Add 4 μl RNase A (100 mg/ml), mix by gentle-vortex or inversion. Incubate for 5 min at room
temperature.
4. Add 200 μl Buffer AL (without adding ethanol). Mix thoroughly by gentle-vortex or inversion.
Incubate at 56°C for 10 min.
5. Add 200 μl of 100% ethanol to the sample. Mix thoroughly by gentle-vortex or inversion.
6. Follow steps 10-14 from tissue sample DNA isolation.
FFPE tissue sections
Use QIAamp DNA FFPE Tissue Kit (Qiagen, cat. #56404) for DNA isolation from FFPE
tissue sections by following procedures in kit’s handbook.
Blood samples
Use DNeasy Blood & Tissue Kit (Qiagen, cat. #69504) for DNA isolation from blood samples
by following blood DNA isolation procedures in kit’s handbook.
DNA quantification and submission
1. Quantify DNA concentration with fluorescent spectrometer with PicoGreen dye (Life
2.
3.
4.
5.
6.
Technologies) or NanoDrop spectrometer.
Purified DNA should show OD260/280 between 1.8 – 2.2 and OD260/230 close to 2.0.
Dilute to 50 ng/l and submit 200 ng – 1.0 g DNA for DNA methylation or SNP array.
Dilute to 100 ng/l and submit 1.0 – 3.0 g DNA for array CGH.
If sample number is larger than 8, submit samples in 8-strip tubes or 96-well microplates.
Submit service request via https://skcccjhmi.corefacilities.org/account/login. (Need to
download sample sheet when making request and upload after filling in sample information).
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Sample preparation and submission guideline
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February 2012