Title: Indirect Genomic Labeling
Pages: 4
Author(s): Claire Reardon
Contact: claire@cgr.harvard.edu
Revision: 1.0 Date: 8/21/03
Reviewers: Christian Daly
Comment: This protocol is based on Pat
Brown’s method which can be found at http://cmgm.stanford.edu/pbrown/mguide/
1. Purpose
This protocol provides instructions for labeling genomic DNA for hybridization to microarrays. The gDNA is labeled with amino allyl dUTP which is then coupled to one of two Cyanine dyes (Cy3 or Cy5). Cy3- and Cy5-labled probes are combined for competitive hybridization to the microarray.
2. Materials
Genomic DNA template(s) (fragmented)
The fragmentation may be done by digesting with DpnII or SauIIA.
Clean up the digested gDNA with a QIAquick purification column before use.
Microarray (blocked and ready to use)
BioPrime Labeling Kit (Invitrogen # 18094011)
Includes 2.5 x primer solution and Klenow Fragment
Nuclease-free water (Ambion # 9932)
100 mM amino allyl dUTP (Sigma Aldrich # A-0410)
100mM dNTPs (Invitrogen # 10297018)
0.5 M EDTA pH 8.0 (Mallinckrodt # 2580)
Microcon YM-30 filters (Millipore # 42410)
1 M NaHCO3 pH 9 (EM Science # SX0320-1)
Cy 3 mono reactive dye pack (Amersham # PA23001)
Cy 5 mono reactive dye pack (Amersham # PA25001)
DMSO 99.9% (Mallinckrodt # 4948)
QIAquick PCR Purification Kit (Qiagen # 28105)
20X SSC (Invitrogen # 15557036)
7 ug/ul poly(A) (Sigma # P9403) or 7 ug/ul poly d(A)–poly d(T) (Sigma #9764)
1 M HEPES pH 7.0 (Calbiochem # 391340)
0.45 µm Ultrafree-MC filters (Millipore # UFC30HVos)
10% SDS (Invitrogen # 24730-020)
Lifterslips (Erie Scientific -Lifterslip size depends on the size of the array)
Staining Dishes with Slide Racks (Thermo Shandon # 121)
3. Instrumentation

Heat block (e.g. VWR standard)
Hybridization chamber (e.g. Telechem # AHC)
Water bath (e.g. Prescision # 5122 1048)
Centrifuge with plate/slide adapter (e.g. Sorvall Legend RT model)
Microcentrifuge (e.g. IEC Micromax RF)
4. Reagent preparation
4.1. 10X aa-dUTP/dNTP mix
4.1.1. Resuspend amino allyl dUTP at 100mM in nuclease free H
2
O.
4.1.2. Mix with stocks of each dNTP for a final ratio of aa-dUTP to dTTP of 2:1.
100mM dATP, dCTP, dGTP
4.8µl (4.8 mM each)
100mM amino allyl dUTP
100mM dTTP
Nuclease Free H
2
O
3.2µl (3.2 mM)
1.6µl (1.6 mM)
90.4µl
100µl
4.2. Cyanine Dyes
4.3.1. Open one dye pack for each dye (Cyanine 3 and Cyanine 5)
4.3.2. Dissolve the contents of each vial in 20 µl DMSO.
4.3.3. Make 10 aliquots of 2µl in 0.5 ml DNAse-free tubes.
4.3.4. Dry the aliquots using a Speed-Vac under low heat for about 1 hour.
4.3.5. Store the dye aliquots protected from light and desiccated at 4 ˚C until use.
4.3. 3x SSC
Dilute the stock of 20x SSC to 3x.
Add 1.5 ml 20x SSC to a 50ml tube.
Add 48.5ml nuclease free water.
4.4. Wash Buffer 1
Milli-Q H2O
20X SSC
10% SDS
4.5. Wash Buffer 2
Milli-Q H2O
20X SSC
774 ml
24 ml
2 ml
798 ml
2 ml
5. Procedure
5.1. Amino Allyl dUTP incorporation
5.1.1. Add 2µg gDNA to a 0.5 ml tube in 24µl nuclease free H
2
O
5.1.2. Add 20µl of the BioPrime 2.5x random primer mix to each reaction
5.1.3. Boil for 5 minutes, then place on ice.

5.1.4. Add 5µl 10x aadUTP/dNTP mix to each tube.
5.1.5. Add 1µl Klenow Fragment (40-50 units/µl) to each tube.
5.1.6. Incubate at 37 o C for 1-2 hours.
5.1.7. Add 5µl 0.5 M EDTA to stop the reaction.
5.2. Cleanup to purify the DNA and remove all Tris before coupling to the dye.
5.2.1. Weigh and label a microcon-30 filter for each reaction.
5.2.2. Dilute each reaction to 500µl by adding 445µl nuclease free water.
5.2.3. Add the mixture to the filter.
5.2.4. Spin at 10,000 xg for 6 minutes and remove the flow through.
5.2.5. Add 470µl nuclease free water
5.2.6. Spin at 10,000 xg for 6 minutes and remove the flow through.
5.2.7. Repeat the rinse from step 5.2.5.
5.2.8. Spin the column until the weight difference is about 18-20 mg.
(18 - 20µl of solution remains on the filter).
5.4.9. Invert the column into a clean tube and collect at 4,500 xg for 5 min.
The volume collected should be 10 – 13µl (the filter retains 5 – 7µl).
5.3. Coupling to the N-hydroxy succinamide ester of the cyanine dye.
5.3.1. Add 1µl NaHCO
3
pH to each sample.
5.3.2. Use the probe resuspend an aliquot of dried cyanine dye (Cy3 or Cy5).
5.3.3. Incubate in the dark at room temperature for 1 hour and 15 minutes.
5.4. Purification and Concentration
5.4.1. Add 70 µl nuclease free H
2
O to each labeling reaction.
5.4.2. Add 500 µl PB buffer.
5.4.3. Apply the mixture to a Qiaquick PCR purification column.
5.4.4. Spin at 13000 rpm for 45 seconds and remove the flow-through.
5.4.5. Add 700 µl PE buffer to the column.
5.4.6. Spin at 13000 rpm for 45 seconds and remove the flow-through.
5.4.7. Repeat this wash from step 5.4.5.
5.4.8. Spin at 14000 rpm for 1 minute to dry the column.
5.4.9. Transfer to the column to a 0.5 ml tube.
5.4.10. Add 30 µl EB buffer to the filter and let it stand for 1 minute.
5.4.11. Spin at 14000 rpm for 1 minute.
5.4.12. Repeat the elution step with another 30 µl EB buffer.
5.4.13. Label and weigh a microcon 30 filter.
5.4.14. Combine the Cy3 and Cy5 labeled probes and add them to the filter.
5.4.15. Spin the filter for 3 minutes at 10,000 xg
5.4.16. Weigh the microcon and concentrate until the desired volume remains.
5.4.16.1. For 16 pin arrays, spin down to 30 mg (30µl).
5.4.16.2. For 32 pin arrays, spin down to 60mg (60µl).
5.4.16.3. For other sizes of arrays, the probe volume must be optimized.
5.4.17. Invert the filter into a clean tube and spin at 4,500 xg for 5 minutes.

5.5. Hybridization Preparation
5.5.1. To the concentrated probe add:
20x SSC
16 pin arrays
3µl poly(A) (7 µg / µl)
1.5µl
1 M HEPES pH 7.0 0.48µl
5.5.2. Wet a Millipore 0.45 µm filter with 10 µl H
2
O.
5.5.3. Spin at 10,000 xg for 1 minute.
32 pin arrays
6µl
3µl
0.96µl
5.5.4. Aspirate the flow-through.
5.5.5. Apply the probe to the filter as a drop.
5.5.6. Spin at 10,000 xg for 1 minute and discard the filter.
5.5.7. Keep the probe at 4˚C until use (up to 12 hours)
5.5.8. Use nitrogen to blow any dust of the microarray
5.5.9. Place the array in a hybridization chamber.
5.5.10. Place a clean Lifterslip over the array with the white strips facing down.
5.6. Hybridization
5.6.1. Add 0.45 µl 10% SDS to the probe.
5.6.2. Heat the probe for 2 minutes at 100˚C.
5.6.3. Allow the probe to cool at room temperatute for 10 minutes
5.6.3.1. Keep the probe protected from light for this time.
5.6.4. Check the volume of the probe.
5.6.4.1. The volume should be 30µl for 16 pin arrays.
5.6.4.2. The volume should be 60µl for 32 pin arrays.
5.6.4.3. For other sizes of arrays, the volume must be optimized.
5.6.4.1. Adjust with water if necessary to achieve the desired volume.
5.6.5. Use a pipet to slowly inject the probe under one corner of the lifter slip.
5.6.6. Move the pipet tip along the edge of the lifterslip as you apply the probe.
5.6.6.1. Make sure not to leave any air bubbles under the lifter slip.
5.6.7. Add 100µl 3x SSC to wells at each end of the hybridization chamber.
5.6.8. Seal the hybridization chamber and place in a 62˚C water bath.
5.6.8.1. Keep chamber level during hybridization.
5.6.9. Allow the hybridization to go for 12–16 hours at 62˚C.
5.7. Washing
5.7.1. Fill two staining dishes with Wash Buffer 1 and two with Wash Buffer 2.
5.7.2. Remove the hybridization chamber from the bath, keeping it level.
5.7.3. Wipe the chamber dry and remove the lid.
5.7.4. Remove the slide from the chamber using forceps.
5.7.5. Submerge the slide in Wash Buffer 1 and allow the Lifterslip to fall off.
5.7.6. Place the slide in a slide rack in Wash Buffer 1 and dunk 20 times.
5.7.7. Transfer the rack to a fresh tank of Wash Buffer 1 and dunk 20 times.
5.7.8. Transfer the slide to clean rack and dunk 20 times in Wash Buffer 2.
5.7.9. Transfer the rack to a clean tank of Wash Buffer 2 and dunk 20 times.
5.7.10. Spin the slides dry at 1000 rpm for 2 minutes.
5.7.11. Store the slides in the dark until they are scanned.
5.7.12. Scan within a few hours of washing.