Probe Labeling, Purification, and Hybridization for MPX 16 Type Microarrays
Version 310506
A. Materials and Reagents
1.) BioPrime DNA Labeling System (Kit), Invitrogen # 18094-011
Contents:
2.5x Random Primers Solution
10x dNTP Mixture (Not Used)
Control DNA (Not Used)
Klenow Fragment
Stop Buffer
Distilled Water
2.) MPX 16 Double-Sided Adhesive Superstructure, Schott Nexterion # 1078355
3.) Nuclease-free water, Ambion, Inc. # 9934
4.) Yeast tRNA, Invitrogen # 15401-011
Add 200ul of 25mg/ml stock to 800ul of nuclease free water to make working solution.
5.) FluoroLink Cy3-dCTP,1mM stocks,Amersham Pharmacia Biotech# PA53021 bulk
pack
PA55321
6.) FluoroLink Cy5-dCTP,1mM stocks Amersham Pharmacia Biotech #PA55021
7.) TE, pH 8.0 (Tris-EDTA)
Add 1ml of 100x TE (Sigma # T-9285) to 99ml of nuclease free water in a culture flask.
8.) 100mM dNTP set, Invitrogen # 10297-018
9.) 10x dNTP mix (For DNA Microarrays)
To 958ul TE add: 12ul dATP, 12ul dGTP, 12ul dTTP, and 6ul dCTP
10.) 20x SSC Solution, Invitrogen # 15557-036
11.) 10% SDS Solution, Invitrogen # 24730-020
Make a 2% solution by adding 1ml of 10% SDS to 4ml of nuclease free water.
12.) 0.1M DTT
Add 0.617g of DTT (Invitrogen # 15508-013) to 40ml DEPC water, store at -20C.
13.) First Wash Solution (Warm to 65C)
880ml distilled water
100ml 20x SSC
10ml 10% SDS
4-5ml 0.1M DTT (Only add immediately before use)
14.) Second Wash Solution
486.5ml distilled water
2.5ml 20x SSC
5ml 10% SDS
5ml 0.1M DTT (Only add immediately before use)
15.) Third Wash Solution
492.5ml distilled water
2.5ml 20x SSC
5ml 0.1M DTT (Only add immediately before use)
16.) Fourth Wash solution
495.75ml distilled water
0.25ml 20x SSC
5ml 0.1M DTT (Only add immediately before use)
17.) Transparency Film, any type,
18.) Strip Tubes and Caps, volume 0.2ml, VWR # 20170-004.
19.) Thermocycler capable of accommodating the above tubes.
20.) PCR Cleanup Plates, Millipore # MSNU03010, OR Microcon YM-30 concentrating
units, Millipore # 42409 or # 24210
21.) Hybridization Incubator, Robbins Scientific # 400.
22.) Water Bath, Precision # 51221048.
23.) Hybridization Containment Tubes, Belco Glass Inc. # AUTOBLOT 200ml.
24.) Binder Clips, Small, UIC # 99020, and medium, UIC # 99050.
25.) Blank, 25x75mm microscope slides.
26.) Printed and Blocked Schott Nexterion MPX16 Slide.
27.) Vacuum Manifold, Multiscreen Filtration System, Millipore # SAVM38401 (only
needed if using PCR cleanup plates- see item 20).
B.) Procedure
Day 1
1.) Add 6ug of each purified, concentrated, and sonicated DNA sample to the 0.2ml
tubes; i.e. 6ug of the comparative DNA and 6ug of the experimental DNA, per array
hybridization. For simplicity, you may want to use one strip tube set for the
experimental, and one strip tube set for the comparative samples.
2.) Add 20ul of the 2.5x Random Primer / Reaction Buffer mix to each tube.
3.) Using a thermocycler program (DNA-MA1), heat these samples for 5min at 100C,
and then cool to 4C until ready for the next step. Remember to activate the hot bonnet.
4.) Add 5ul of the 10x dNTP mix (For DNA Microarrays) to each tube. Do not use the
dNTP mix supplied with the BioPrime DNA Labeling Kit, as this contains biotinilated
nucleotides.
5.) Add 1ul of the Cy5-dCTP or Cy3-dCTP (1mM stocks) to their respective reaction
tubes.
6.) Add 1.5ul of the Klenow fragment.
7.) Using a thermocycler program, incubate the samples at 23C for 16 hours.
Day 2
8.) Prepare the slide-superstructure complex as follows. Expose the adhesive on one side
of the superstructure by removing the protective paper barrier. Carefully align the
superstructure above the printed and blocked slide, and lightly place onto the slide.
Check and ensure that the superstructure is only in contact with the black patterning
material of the slide; if not make adjustments. Firmly bond the superstructure to the slide
with the use of a small roller. Firmly roll across the superstructure at least 45 times, in
parallel with each of its four sides. Do not press down so hard so as to warp the
superstructure.
9.) Using a standard 25x75mm slide as a guide, cut a piece of plastic transparency for
use as a cover for the slide complex. Wash this “cover” by shaking in a tube filled with
50ml of distilled water, and another filled with 100% ethanol. Let this dry by hanging
from a binder clip.
10.) Turn on the incubator, and set its temperature to 65°C.
11.) Stop the labeling reactions by adding 5ul of the Stop Buffer to each tube.
*Steps 12-16 in this protocol can be substituted with Steps 5-12 of the DNA labeling AL
slide protocol (i.e., using YM30 microcons to wash and concentrate probe, rather than a
vacuum filter-plate cleanup system).
12.) Pipette these samples into appropriate wells of the Millipore filter plate, .i.e. occupy
one of the plate’s columns with the contents of one of the strip tube complexes.
13.) Add 200ul of nuclease free water to each well containing a sample.
14.) Place the plate onto the vacuum manifold, and apply the vacuum to between -20 and
-25 inHg. Once liquid levels within the wells are lower, add another 200ul of nuclease
free water. Continue washing thru water until a total of 1000ul has been added to each
well. Always keep enough water in the wells to avoid drying the sample against the filter
surface. Once the last aliquots have been added, reduce the sample volume to 50>100ml.
15.) Turn the vacuum off. You will note that the labeled probe tends to collect onto the
filter surface. Re-suspend the probe using repeated pipette siphoning. Tilt the plate to a
45º angle, place the pipette’s tips lightly against the bottom edge of the wells, and remove
the samples. Combine the appropriately labeled Cy3 and Cy5 samples into fresh filterplate wells, and add 20ul of yeast t-RNA to each.
16.) Add 200ul of nuclease free water to each well and repeat the filtering procedure
described above, except filter through a total of only 400ul.
17.) The final sample volume should be reduced to 16ul.
18.) Remove the samples as described before, and place into labeled strip tubes.
19.) To each combined sample add: 11.5ul 20xSSC
these can be pooled
2.8ul Yeast tRNA
16 uL Nucl. free H2O
2.8ul 2% SDS
0.54ul 0.1M DTT
20.) Using a thermocycler program (DNA-MA3), heat each sample to 100C for
1.5min, and then incubate for 37C until ready for array application.
21.) During this incubation period, clean the array surfaces of the superstructure-slide
complex by carefully flushing with a stream of an inert gas.
22.) Remove the remaining adhesive protector from the superstructure.
23.) After the incubation, carefully pipette 50ul of the purified labeled probe onto the
appropriate array chambers of the superstructure-slide complex. You may choose to use
a multi-channel pipette.
24.) Carefully align and place the plastic transparency strip onto the superstructure.
Tightly bond this cover by smoothing a roller over its surface. Repeatedly role over the
surface at least 50 times in each horizontal and vertical direction. Do not press so hard as
to warp the superstructure. Finally, place a 25x75mm slide over the superstructure and
secure it with two small binder clips at each end, and a medium binder clip in the center.
Remove, and save, the medium binder clip handles.
25.) Place the superstructure-slide complex into a hybridization containment tube.
Ensure that it does not move about within this tube by securing with packed aluminum
foil. Place the screw cap on top, and place the tube into the pre-heated incubator. Rotate
at 12rpm.
26.) Prepare the First Wash Solution (without DTT) and place overnight in a 65ºC water
bath.
Day 3
27.) Prepare wash solutions 2, 3, 4 and place them into separate wash tanks. Remove
the first wash solution from the 65°C water-bath and pour 500ml into a wash tank, and
approx 400ml into a squeeze bottle.
28.) Add 4-5 ml DTT to each wash tank solution, but not the squeeze bottle.
29.) Remove superstructure-slide complex from the hybridization oven, and remove the
clips and the slide support. Remove the plastic transparency strip by carefully pealing it
back from the superstructure using sharp-tipped forceps. Be careful not to scratch the
slide surface, or to remove the superstructure from the slide at this point. Immediately
plunge the complex face-up into the first tank containing the First Wash Solution.
30.) Using the squeeze bottle, expel a strong stream of the First Wash Solution into each
of the array wells while holding the complex at an angel above an empty tank. Repeat
this several times for each array-well. Concentrate on cleaning the corners of the wells
since this is where debris tends to accumulate. Never allow the array wells to dry.
31.) Immerse the complex into the wash tank with the residual First Wash Solution to fill
completely each well. While the complex is faced up, remove it from the tank and place
onto a paper towel. Carefully remove the superstructure from the slide with the same
pointed forceps, being careful not the scratch the slide.
32.) Place a slide rack in the fresh First Wash Solution tank and immediately place this
slide into the rack. Further wash the slide in this tank for 5min, while swirling the rack
vigorously so as to remove any additional debris from the slide’s surface.
33.) Move the slide rack to the Second Wash Solution, and repeat the previous washing
procedure.
34.) Move the slide rack to the Third Wash Solution, and repeat the washing sequence
for 2min.
35.) Move the slide rack to the Fourth Wash Solution, and repeat the washing sequence
for 2 min.
36.) Quickly dry the washed slides via centrifugation at 250 x g for 2.5 min.
37.) Scan the slides as soon as possible.