Genomic DNA Labeling Protocol for Genotyping Microarrays
Version 053106
A. Reagents (1 – 10 and 13 are nuclease-free)
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 (Not Used)
2.) Nuclease-free water, Ambion, Inc. # 9934
3.) Yeast tRNA, Invitrogen # 15401-011
Add 200ul of 25mg/ml stock to 800ul of nuclease free water to make working solution.
4.) FluoroLink Cy3-dCTP,1mM stocks,Amersham Pharmacia Biotech# PA53021 bulk
pack
PA55321
5.) FluoroLink Cy5-dCTP,1mM stocks Amersham Pharmacia Biotech #PA55021
6.) TE, pH 8.0 (Tris-EDTA)
Add 1ml of 100x TE (Sigma # T-9285) to 99ml of nuclease free water in a culture flask.
7.) 100mM dNTP set, Invitrogen # 10297-018
8.) 10x dNTP mix (For DNA Microarrays)
To 958ul TE add: 12ul dATP, 12ul dGTP, 12ul dTTP, and 6ul dCTP
9.) 20x SSC Solution, Invitrogen # 15557-036
10.) 10% SDS Solution, Invitrogen # 24730-020
Make a 2% solution by adding 1ml of 10% SDS to 4ml of nuclease free water.
11.) 0.1M DTT
Add 0.617g of DTT (Invitrogen # 15508-013) to 40ml DEPC water, store at -20C.
12.) 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)
13.) Second Wash Solution
486.5ml distilled water
2.5ml 20x SSC
5ml 10% SDS
4-5ml 0.1M DTT (Only add immediately before use)
14.) Third Wash Solution
492.5ml distilled water
2.5ml 20x SSC
4-5ml 0.1M DTT (Only add immediately before use)
15.) Fourth Wash solution
495.75ml distilled water
0.25ml 20x SSC
4-5ml 0.1M DTT (Only add immediately before use)
16.) 4x SSC
8ml distilled water.
2ml 20x SSC
17.) Thin-Wall PCR Tubes, 0.5ml, MJ Research # TBI-0501
18.) Thermocycler capable of accommodating the above tubes.
19.) Microcon YM-30 concentrating units, Millipore # 42409 OR # 24210
20.) Slide Hybridization Chambers, Telechem International Inc # AHC-1
21.) Water bath suitable for containment of the above chambers.
22.) Lifter (cover) Slips, Erie Scientific Company # 22X25I-2-4635.
B.) Procedure
DAY 1:
1.) Add 10ul of 600ng/ul DNA (i.e. 6ug) of each purified, concentrated, and sonicated
DNA sample to its own 0.5ml thin-walled PCR tube; i.e. 6ug of the comparative DNA
and 6ug of the experimental DNA, per array co-hybridization.
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.) On ice, 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 1.5ul of the Cy5-dCTP or Cy3-dCTP (1mM stocks) to their respective reaction
tubes.
6.) Add 2ul of the Klenow fragment.
7.) Using a thermocycler program (23 deg), incubate the samples at 23C for 16 hours.
DAY 2:
1.) Fill the water bath, and set its temperature setting to 65°C.
2.) For each array (combined sample), weigh a Microcon 30 filter and set aside.
3.) Clean (with EtOH or compressed CO2) the hyb chambers you need for your
experiment. Place the printed and blocked slides onto the platform of the hybridization
chamber, ensuring that the DNA printed side is facing upwards. Always handle the slides
and slips by their edges, and while wearing powder free gloves. Select a “lifter” slip,
determine the side containing the lifters, and clean this surface using an inert compressed
gas. (CAUTION! Always wear safety glasses during this step). Place the slip onto the
surface of the array, ensuring that the lifters are in contact with the slide, and that the
DNA arrayed area is completely covered. Handling may be facilitated with the use of a
vacuum pen. Clean (with EtOH or compressed CO2) the top of the hyb chamber and
place cover the slides and slips, to stop dust etc falling in, whilst you prepare the rest of
the experiment.
4.) Stop the reactions by adding 5ul of the Stop Buffer to each tube.
5.) Add 450ul of nuclease free water to each reaction tube.
6.) Add this solution to a Microcon 30 filter. Centrifuge the filter at 8000 x g for 10min.
Remove the filtrate, and repeat steps 5 and 6.
7.) Invert the filter into a clean collection tube, and centrifuge at 8000 x g for 2min. The
collection should consist of 20-40ul.
8.) Combine the appropriate Cy5 and Cy3 labeled samples, and then add:
20ul Yeast t-RNA
450ul Nuclease-free water
9.) Add each combined sample to the appropriately labeled Microcon 30 filter from step
#2.
10.) Initially, centrifuge the filters at 8000 x g for 13min.
11.) Weigh the sample containing filters to determine the remaining volumes (1ul = 1ug)
12.) Continue to concentrate the samples until about 12ul remains. Recover samples as
before by inverting filter into a clean collection tube, and centrifuging at 8000 x g for
2min. Add nuclease free water if the liquid volume falls below 12ul (NB: 16ul if using
MPX slides).
13.) Take clean thermocycler tubes and add: To each combined sample add:
8.5ul 20xSSC
these can be pooled
2.1ul Yeast tRNA
12ul Nuclease Free Water
12uL DNA
0.4ul 0.1M DTT
2.1ul 2% SDS
14.) Using a thermocycler program (DNA-MA3), heat each sample to 100C for 1.5min,
and then incubate for 37C until ready for use.
15.) Remove the sample tubes from the thermocycler, and place them into the microfuge
centrifuge. Pulse to bring down condensation.
16.) Make sure the lifter slips are aligned accurately etc (use fine forceps). Carefully and
slowly pipette approx 37ul onto the appropriately labelled arrays. The samples are
pipetted at the open interface of the lifter slip and array slide. Do not touch the slide or
cover slip with the pipette tip. Capillary action will spread the sample evenly throughout
the array surface.
17.) Add 100ul of 4x SSC to each of the two recessed wells of the hyb chamber, for a
total of 200ul per chamber.
18.) Tightly cover the hybridization chambers, and GENTLY place into the 65C
waterbath. Incubate overnight, and ensure that they are not exposed to light.
19.) Prepare warm wash# 1 and place in a water bath to 65C overnight. Take aliquot of
DTT (16- 20ul) from the freezer and store in the fridge overnight.
DAY 3:
20.) Prepare 5 basins for the wash solutions (2 basins for wash#1 solution). Add 500ml
of wash solution to each basin, but only 200 ml to the 1st basin of wash#1 solution, and
300 ml of wash#1 solution in a squeeze bottle.
21.) Add 4 – 5ml DTT to 4 basins (omit adding DTT to the 1st basin of wash#1).
22.) Place an empty slide rack in the 2nd basin of wash#1. Also prepare a centrifuge with
slide rack carriers. Place a slide rack with balancing mock slides on one of these carriers.
Set the centrifuge to 250 x g, 2.5 min, and 22°C.
23.) Remove array slides from the hybridization chambers, and remove the cover-slips by
plunging the array slides (inverted at 45º angle) into the 1st basin of wash#1. Using the
squeeze bottle, expel a strong stream of the First Wash Solution onto each of the array
wells while holding the slide at an angel above the tank (1st basin of wash#1). Repeat this
several times for each array. Do not let slide dry out. Place the slide into the slide rack in
the 2nd basin of wash#1. Repeat for all slides. Once all the slides are in the rack, plunge it
up and down repeatedly (45 times) in the wash for 5min.
24.) Move the slide rack to the Second Wash Solution, and repeat the previous washing
procedure.
25.) Move the slide rack to the Third Wash Solution, and repeat the washing sequence
for 2min.
26.) Move the slide rack to the Fourth Wash Solution, and repeat the washing sequence
for 2min.
27.) Quickly dry the washed slides via centrifugation.
28.) Scan the slides as soon as possible.