JHU Oncology Microarray Facility
DNA Preparation Protocol and Submission Guideline
Core uses and recommends DNeasy Blood & Tissue Kit (Qiagen) for genomic DNA
preparation. Look in kit’s handbook for details.
Tissue samples
1. Cut up to 25 mg tissue into small pieces under frozen condition and place in a 1.5 ml
microcentrifuge tube.
2. Add 180 l Buffer ATL and 20 l proteinase K provided in the kit.
3. Mix thoroughly by gentle-vortexing or inversion.
4. Incubate at 56oC until the tissue is completely lysed (overnight lysis is recommended).
5. Gentle-vortex occasionally during incubation.
6. Add 4 l RNase A (100 mg/ml), mix by gentle-vortexing or inversion and incubate for 5
min at room temperature.
7. Add 200 μl Buffer AL to the sample and mix thoroughly by gentle-vortexing or
inversion.
8. Add 200 μl of 100% ethanol and mix thoroughly by gentle-vortexing or inversion.
9. Pipet the mixture (including any precipitate) into the DNeasy Mini spin column placed
in a 2 ml collection tube. Centrifuge at 6000 x g (8000 rpm) for 1 min. Discard flowthrough and collection tube.
10. Place the DNeasy Mini spin column in a new collection tube, add 500 μl Buffer AW1,
and centrifuge for 1 min at 6000 x g (8000 rpm). Discard flow-through and collection
tube.
11. Place the DNeasy Mini spin column in a new collection tube, add500 μl Buffer AW2,
and centrifuge for 3 min at 20,000 x g (14,000 rpm) to dry the DNeasy membrane.
Discard flow-through and collection tube.
12. Place the DNeasy Mini spin column in a microcentrifuge tube, and pipet 100 μl Buffer
AE directly onto the DNeasy membrane. Incubate at room temperature for 1 min, and
then centrifuge for 1 min at 6000 x g (8000 rpm) to elute DNA.
13. If maximum yield is preferred, elution can be done with two successive steps of adding
200 ul nuclease-free water in each step and concentrate the combination by speedvacuum.
Cultured cells
1. Centrifuge the appropriate number of cells (5x 105 – 2x 106) for 5 min at 300 x g.
Resuspend the pellet in 200 μl PBS. Add 20 μl proteinase K.
2. Add 4 μl RNase A (100 mg/ml), mix by gentle-vortexing or inversion, and incubate for
5 min at room temperature.
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Sample preparation and submission guideline
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February 2012
JHU Oncology Microarray Facility
3. Add 200 μl Buffer AL (without adding ethanol). Mix thoroughly by gentle-vortexing or
inversion, and incubate at 56°C for 10 min.
4. Add 200 μl of 100% ethanol to the sample, and mix thoroughly by gentle-vortexing or
inversion.
5. Pipet the mixture into the DNeasy Mini spin column placed in a 2 ml collection tube.
Centrifuge at 6000 x g (8000 rpm) for 1 min. Discard flow-through and collection tube.
6. Place the DNeasy Mini spin column in a new collection tube, add 500 μl Buffer AW1,
and centrifuge for 1 min at 6000 x g (8000 rpm). Discard flow-through and collection
tube.
7. Place the DNeasy Mini spin column in a new collection tube, add 500 μl Buffer AW2,
and centrifuge for 3 min at 20,000 x g (14,000 rpm) to dry the DNeasy membrane.
Discard flow-through and collection tube.
8. Place the DNeasy Mini spin column in a microcentrifuge tube, and pipet 100 μl Buffer
AE directly onto the DNeasy membrane. Incubate at room temperature for 1 min, and
then centrifuge for 1 min at 6000 x g (8000 rpm) to elute DNA.
9. If maximum yield is preferred, elution can be done with two successive steps of adding
200 ul nuclease-free water in each step and concentrate the combination by speedvacuum.
RNA quantification and submission
1. Measure DNA concentration with fluorescent spectrometer with PicoGreen dye or
NanoDrop.
Purified DNA should show OD260/280 between 1.8 – 2.2 and OD260/230 close to 2.0.
Dilute to 50 ng/ul and submit 1 – 2 ug DNA for DNA methylation or SNP array.
Dilute to 100 ng/ul and submit 3 – 4 ug DNA for array CGH.
If sample number is larger than 8, submit samples in 8-strip tubes or microplate.
Fill service request form and sample sheet downloadable from Core’s website,
http://microarray.onc.jhmi.edu.
7. Send electronic version of sample sheet to the Core.
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Sample preparation and submission guideline
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JHU Oncology Microarray Facility
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Sample preparation and submission guideline
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February 2012