Shirley Zhu/ Genomic DNA labeling
Array-Based Comparative Genomic Hybridization Protocol
(Based on the protocol from Pollack lab)
Digestion and Purification of Genomic DNA
- 4 ug of DNA from sample (adjust volume accordingly for each sample), add 1 ul of DpnII, 4ul of 10X DpnII buffer, up to 40ul of total volume. (Water and DNA together should be 35 ml; then heat it at 60/55°C for 10 min; then add DpnII and the buffer. This helps dissolve DNA in water well.) Mix well.
- Digest for 1.5 hours at 37 C
- Inactivate by heating at 65 C for 20 min, then snap cool on ice Add 60ul of TE (ph8.0) to each sample to bring volume up to 100ul
- Add 500ul (5 volume) of Buffer PB (from QIAGEN, QIAquick PCR Purification Kit, CAT#28194), and invert several times before a quick spin
- Apply 600ul to column, spin for 30 seconds, discard flow-thru
- Add 750ul of Buffer PE and spin for 30 second
- Spin additional 1 min to dry column
- Place column into a new eppendorf tube
- Add 23ul 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. RCF to recover bound DNA
Labeling of Genomic DNA Fragments
-Transfer the 21-23ul of DNA to a micro centrifuge tube
-Add 20ul of 2.5 X random prime mix( Invitrogen, BioPrime DNA Labeling System Kit, CAT# 18094-011) to 21-23ul of DNA
-Boil 5 min, then place on ice for 5 min, do a quick spin at 4 C
-Mixture: DNA 41ul
10X dCTPdNTP mix 5ul
Cy3/Cy5 3ul
Klenow 1ul (from the Kit)
Total volume 50 ul
- Tap gently and give it a quick spin
- Incubate 2 hours at 37C
- Add 5 ul of stop solution( from the Kit)and put back on ice.
Concentrating Labeling Probes and hybridization to arrays
-Combine Cy3 and Cy5, put Cy3 to Cy5
-Add 400 ul of TE (pH8.0) to each combined probe
-Apply the labeled probe to a Microcon YM30 (Millipore, Cat. No.: 42410)
-Spin at 12,000xg for 11 min, make the volume less than 20 ul or so(½ of the filter is visible),
-Discard flow-thru, wash with additional 500 ul TE (pH8.0), and spin at 12,000xg for 11 min, make the volume less than 20 ul
-Make mix: Human Cot-1 DNA 50 ul
Yeast tRNA 20 ul( 5ug/ul)
Poly dT-dA 4 ul(5ug/ul)
TE (pH8.0) 450ul
Total about 520 ul
-Mix well, add to the filter
-Spin for 11-12 min at 12,000xg, make the volume less than 32 ul
-Invert the filter to a new tube, spin 2 min to recover the labeled probe
-Adjust the volume by Dnase free water to 32 ul
-Then make: Probe 32 ul
20X SSC 6.8 ul
10 % SDS 1.2 ul
Total volume 40 ul
( wipe off additional SDS on pipette tip with side of gloves)
-Mix lightly to minimize bubbles, and give a quick spin
-Denature probe(s) by heating for 2 min at 100 C, then quick spin
-Incubate at 37C for 30 min for Cot-1 to block repetitive DNA in sample
-Pre-warm Hyb charmbers on top of 65 C Hyb water bath
-Spin DNA probe 5 min at max rcf
-Working quickly , place 40 ul of probe onto array
-Add 20 ul of 3X SSC drops scattered evenly across the top of the cover slip. Tighten screws and incubate at 65 C overnight.
Wash arrays as with mRNA labeling protocol and scan:
-Then transfer the slides to 2X SSC/0.03%SDS at 65C, shake the slide holder up and down vigorously for 5 min, making sure slides never out solution.
-Wash slides in 2XSSC for 5 min, same as above or with a shaker
-Wash slides in 1XSSC for 5 min, same as above
-Wash slides in 0.2XSSC for 5 min, twice
-Spin slides down in centrifuge at 500 rpm for 5 min (prepare everything in advance, so transfer of slide rack to holder is very quickly done)
- Clean box needed to transport slides to scanner
-Scan immediately!
Washing buffer:
20X SSC stock solution 10% SDS
500 ml of 2X SSC/0.03% SDS 50 ml 1.5 ml at 65 C
500 ml of 2X SSC 50 ml
500 ml of 1X SSC 25 ml
500 ml of 0.2 X SSC 5 ml
Note:
The first washing step should be performed at 65° C; this appears to significantly increase the specific to non-specific hybridization signal.
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