Cy-Labeling of Genomic DNA using Random-Priming
Labeling Reaction
Set up labeling reactions in duplicate, you will need one reaction for the Cy-5 labeling and another one for Cy-3 labeling reaction.
2.0 microg genomic DNA Sheared to ~500bp
2.5 microL pd(N)6 Random Hexamer at 50 OD U/mL (about 12 microg)
Add water to a final volume of 23 microL
Heat to 100 degrees C for 2 minutes to denature DNA.
Cool down tubes on bench.
Spin Down condensation
To each of the denatured tubes add: (label one tube Cy-3 and the other Cy-5)
0.5 microL 100X dNTP mix
1.5 microL Cy-3 or Cy-5 (add one or the other, depending on the reaction)
16 microL ddH2O
5 microL 10X Klenow Buffer (Comes with enzyme)
4 microL 3'-5' exonuclease-free Klenow fragment 5U/ microL (NEB cat# 212)
Final volume 50 microL
Incubate at 37 degrees C for 2 hrs
Spin down condensation
Stop reaction by adding 5 microL 0.5M EDTA pH 8.0, mix well
Purification of Probes
1. Add 450 microL TE pH 7.4 to each stopped labeling reaction.
2. Gently pipet onto Microcon 30 filter
3. Spin ~10 min at 8000g (10,000 rpm in microcentrifuge).
4. Invert and spin 1 min at 8000g to recover purified probe to new tube (~20-40 microL volume). Be aware that the inverted column only fits on the tubes provided by Millipore with the filters, it does not fit regular microcentrifuge tubes.
5. For two-color array hybridizations, combine purified probes (Cy5 and Cy3 labeled probes) in new microcentrifuge tube
6. Then add:
10 microL Sheared Salmon Sperm DNA at 10mg/mL
450 microL TE pH 7.4
7. Concentrate with a Microcon 30 filter as above (8000g, ~15 min, then check volume every 1 min until appropriate).
8. Collect probe mixture in a volume of 24 microL or less.
Hybridization
1. Adjust volume of probe mixture to 24 microL with ddH20.
2. Add 5.1 microL 20X SSC (for a final concentration of 3.4X)
3. Add 0.9 microL 10% SDS (for a final conc. of 0.3%).
Note: In this case the final volume of hybridization is 30 microL. Volumes should be adjusted up or down according to the size of the cover slip
4. Denature hybridization mixture (100 degrees C, 1.5 min)
5. Incubate for 30 minutes at 37 degrees C
6. Place probe gently on the spotted area of the slide. Gently lowered coverslip until it adheres to the slide.
7. The microarray was then placed in a waterproof hybridization chamber, hydrated with a drop of water on both ends of the slide, away from the coverslip, for hydration.
8. Seal chamber and immerse slide in a 65 degrees C waterbath
9. Hybridize microarray at 65 degrees C overnight (16-20 hrs).
Washing
1. After hybridization, remove the hybridization chambers from the 65 degrees C waterbath, dry off, and open.
2. Place microarray slides in glass slide racks, and immerse in slide chamber with 200mL of 2X SSC, 0.03% SDS in it.
3. Gently agitate until coverslip slid off
4. Incubate at 65 degrees C for 5 minutes
5. Remove slide rack, place over paper towels to gently dry.
6. Immerse slide rack in slide chamber with 200mL of 1X SSC in it. Incubate five minutes at room temperature
7. Remove slide rack, place over paper towels to gently dry.
8. Immerse slide rack in slide chamber with 200mL of 0.2X SSC in it. Incubate five minutes at room temperature.
9. Dry slides by centrifuging the glass rack in a table top centrifuge designed to hold 96-well plates for 3-4 minutes at setting 600-700 rpm.
10. Store slides in the dark until scanning.
Note: A)The first washing step should be performed at 65 degrees C; this appears to significantly increase the specific to non-specific hybridization signal.
B)If you are processing only one slide 50mL conical tubes work well as chambers.