HYBRIDIZATON PROTOCOLS:
Each hybridization should have the appropriate blocking agents added. Some include
the following at the indicated final concentrations:
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Poly-A( 40-60) ~0.4ug/ul (blocks oligo-dT used in labeling reaction)
PolydT ~0.4ug/ul (blocks PolyA tails of mRNA)
Yeast tRNA ~0.4ug/ul ( blocks nonspecific DNA hybridization)
Cot-1 DNA ~10 to 50ug total (organism specific available, blocks hybridization
to repetitive DNAs )
Denhardts Solution ~1-5x final ( BSA, Ficoll, PVP)
Others add additional buffering agents such as HEPES to a final concentration of 50mM,
or change the SSC to SSPE. I find SSC sufficient for most applications.
I will refer to the solutions below as “hyb solutions”, and to the solutions with added
blockers as “hyb cocktail”.
Coverslip Option 1:
(Hybridization to arrays spotted with PCR products)
50% Formamide, 3x SSC, 0.1% SDS, +blocking agents
Hyb at 42C
Coverslip Option 2:
(Hybridization to arrays spotted with long Oligos)
25% Formamide, 3x SSC, 0.1% SDS, +blocking agents
Hyb at 42C
Coverslip Option 3:
(Hybridization to arrays spotted with long oligos)
4x SSC, 0.1%SDS, +blocking agents
Hyb at 65C
Agilent hyb chamber Option 1:
(Hybridization to in house long oligo arrays)
1x In-situ buffer, 1x Blocking Agents
Hyb at 60C in rotating oven.
Agilent hyb chamber Option 2:
Formamide Based Hybridization Buffer
Agilent 10X Blocking Agent
100% Formamide
10% Dextran Sulfate
20X SSC
10% SDS
Cy3 and Cy5 Sample + dH2O to
40 µl
120 µl
141 µl
80 µl
8 µl
11 µl
(1x )
(30%)
(3.525%)
(4x)
(0.2%)
400 µl
Hybridize in Rotating Oven @ 42°C for 17-20 hours
For most arrays I recommend adding a balanced amount of nucleic acid as
measured post dye incorporation. Depending on the complexity of the array/
organism, this amount may vary. QC your dye incorporation prior to using
your sample on the Nanodrop to make sure you will have enough dye
incorporation. Corning has general recommendations based on the initial
material labeled. If you start with total RNA, use a final minimum amount
of 1.0 pmole/ul/dye, mRNA use 0.25 pmole/ul/dye, CGH DNA use 1.0
pmole/ul/dye. Adding too much can be as detrimental as adding too little,
and these amounts vary by organism.
So, for example, you started with total RNA, if your weaker incorporation
sample requires 6 ul to have 40 pmoles of incorporated material in a hyb
volume of 40 ul (1 pmole/ul final and this corresponds to 1.3ug of nucleic
acid for that channel, Add 1.3ug of the other sample. Remember the
differences in dye incorporation will be washed out in data normalization
down stream.
Typically these amounts are in volumes that are workable to add a 2x hyb
cocktail to and bring up to final volume with water. If the volume becomes
difficult, speedvac to a manageable volume, or to just dry, then add cocktail.
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Prepare sample in appropriate volume of Hyb cocktail for the
coverslip size, + ~3ul. This extra will be left in the tube with any
potential large debris later.
Heat to 95C for 5 min.
Vortex well, cool RT 2 min.
Quick high-speed spin to remove bubbles and bring large fragments to
the bottom of the tube.
Draw the appropriate amount for the coverslip leaving the extra
volume and any potential chunks at the bottom of the tube.
Load the array by pipetting from one end of the lifterslip and letting it
wick under until full.
Pipette extra blank hyb solution (-blockers) into the appropriate
hydration locations for your hyb chamber.
Seal the chamber and incubate in a water bath at the appropriate hyb
temperature for your buffer.
Incubate typically 16-20 hours.
For Agilent buffer and gasket cover slips, we recommend calculating based
on a ~60ul base hyb, even though the final volumes will be 200ul or 450 ul.
Materials:
Formamide
SSC
SDS
Hyb chambers (Monterey Industries)
http://www.montereyindustries.com/dualhybridchamber.htm
Lifterslips, M-series (Erie Scientific)
http://www.eriemicroarray.com/coverglass/lifterslips-m.aspx
Poly-A (40-60)
PolydT
Yeast tRNA
Cot-1 DNA
Denhardt’s Solution
(Sigma or your favorite supplier)
OR
Agilent gasket slides/coverslips
->2x model G2534-60002 (5pack) or G2534-60006( 100pack)
->1x model G2534-60003(5pack) or G2534-60005(100pack)
2x In-situ hyb buffer (Agilent 5185-5973)
10x blocking agents ( Agilent 5188-5281)