Shirley Zhu aCGH_Agilent
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Probe labeling and hybridization on Agilent Oligo ArrayBased CGH
(Agilent labeling kit Cat# 5188-5309, 5188-5220)
Modified by Shirley Zhu
Restriction Digestion (using micro tubes)
Genomic DNA
4μg (option: heat the DNA at 55/60C for 10min before using)
Nuclease-free water
up to volume
20.2ul
-Preparation of Digestion Master Mix (for 4x44K microarrays)
Component Per reaction
(μl)
10X Buffer C*
2.6
Acetylated BSA (10μg/μl)*
0.2
Alu I (10 U/μl)
1.5
Rsa I (10 U/μl)
1.5
Final volume
5.8
*Supplied with the restriction enzyme Rsa I.
-Add 5.8μl of Digestion Master Mix to the genomic DNA
-make a total volume of 26 μl
-Mix well by pipetting up and down.
-Incubate at 37°C for 2 hours.
-Incubate at 65°C for 20 minutes to inactivate the enzymes.
-Move the sample tubes to ice.
- Add 74ul of TE (ph8.0) to each sample to bring volume up to 100ul
-Add 500ul (5 volume) of Buffer PB (QIAGEN MinElute PCR Purification Kit CAT#
28004), mix without vortex
-Apply 600ul to column, spin for 1 min at max speed, and discard flow-through
-Add 750ul of Buffer PE and spin for 1min
-Spin additional 3 min to dry column
-Place column into a new eppendorf tube
-Add 14ul of TE (ph8.0) directly on top and center of the filter
-Let it sit at RT for 1 min
-Spin 1 min at max. Speed
-Add another 14ul of TE (ph8.0) directly on top and center of the filter
-Spin 1 min at max. Speed again
- To recover bound DNA with total volume of 28ul about, adjust the volume by nucleasefree water
-using 1.5ul for Nano drop to measure OD
-leave 26ul for labeling.
-Proceed directly to Sample Labeling or store digested genomic DNA overnight
at -20°C.
Shirley Zhu aCGH_Agilent
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Fluorescent Labeling of DNA
-Add 5μl of Random Primers (supplied with Agilent Genomic DNA Labeling
Kit PLUS) to each reaction tube containing 26μl of digested gDNA to make a total
volume of 31 μl
-Mix well by pipetting up and down gently.
-Incubate at 95°C for 3 minutes
-move to ice and incubate on ice for 5 minutes.
-Preparation of Labeling Master Mix:
Component Per reaction
(μl)
5X Buffer
10.0
10X dNTP
5.0
Exo-Klenow fragment
1.0
Final volume
16.0
-add Cyanine 3-dUTP (1.0 mM) to Ref DNA
-add Cyanine 5-dUTP (1.0 mM) to Samples
3ul or
3ul
Total volume:
19ul
-Add 19 μl of Labeling Master Mix to 26ul of digested gDNA,
-make a total volume of 50μl.
-Mix well by gently pipetting up and down.
-Incubate at 37°C for 2 hours.
-Incubate at 65°C for 10 minutes to inactivate the enzyme, then move to ice.
Clean-up of Labeled Genomic DNA
-Combine the 50ul of cyanine 5-labeled sample and 50ul of cyanine 3-labeled
Sample, get 100ul of mixture samples.
-Add 450μL of 1X TE (pH 8.0) to a Microcon YM-30 filter (the filter is into a 1.5-ml
microfuge tube, supplied).
-load each labeled gDNA into the filter, mix well
-Spin 10 minutes at 12,000 × g in a micro centrifuge at room temperature. Discard the
flow-through.
-Add 450μl of 1X TE (pH 8.0) to each filter. Spin for 11 minutes at 12,000 × g
in a microcentrifuge at room temperature. Discard the flow-through.
-Add 450 μl of 1X TE (pH 8.0) to each filter. Spin for 11 minutes at 12,000 × g
in a microcentrifuge at room temperature. Discard the flow-through.
-Invert the filter into a fresh 1.5-mL microfuge tube (supplied).
-Spin for 1minute at 12,000 × g in a microcentrifuge at room temperature to collect
purified sample.
-Measure and record volume (μl) of each eluate. If sample volume exceeds
40μL, return sample to its filter and spin 1 minute at 8,000 × g in a
microcentrifuge at room temperature. Discard the flow-through.
-Spin until each sample volume is ≤ 40μL.
Shirley Zhu aCGH_Agilent
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-Bring total sample volume to 40μL with 1X TE (pH 8.0).
-Take 1.5μl of each sample to determine the yield and specific activity by
using the NanoDrop ND-1000 UV-VIS Spectrophotometer..
-Labeled DNA can be stored overnight at -20°C in the dark.
Preparation of Labeled Genomic DNA for Hybridization
-Prepare the 10X Blocking Agent:
a. Add 1350μl of nuclease-free water to the vial containing lyophilized 10X
Blocking Agent (supplied with Agilent Oligo aCGH Hybridization Kit).
b. Leave at room temperature for 60 minutes and mix on a vortex to
reconstitute sample before use or storage.
The 10X Blocking Agent can be prepared in advance and stored at -20°C.
Assembly of hybridization samples:
Component
Volume (μl) per hybridization
-Cyanine 5- and cyanine 3-labeled gDNA mixture
39
-Human Cot-1 DNA (1.0 mg/ml)
5
-Agilent 10X Blocking Agent
11
(Supplied with Agilent Oligo aCGH Hybridization Kit)
-Agilent 2X Hybridization Buffer
55
Final Hybridization Sample Volume
110
-Mix the sample by pipetting up and down, then quickly spin in a
microcentrifuge to drive contents to the bottom of the reaction tube.
-Incubate at 95°C for 3 minutes.
-Immediately Incubate at 37°C for 30 minutes.
-Spin 1 minute at 13000rpm in a microcentrifuge to collect the sample at the bottom of
the tube.
-The DNA probe is ready for hybridization