IGF APEX PROTOCOL
PCR amplification:
The PCR reaction in total volume of 30 l will contain:
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Sterile deionised water (MQ)
10x Taq DNA polymerase buffer (1X at final concentration)
2.0 mM dNTP (20% dUTP) (0.20 mM at final concentration)
50 mM MgCl2 (2.5 mM MgCl2 at final concentration)
10 pmol/µl F+R primer stock (20 pmol of each primer pair)
DNA (17 ng per reaction minimum)
Platinum Taq DNA polymerase (5U/ l stock, i.e. 0.6U per reaction)
~20 l
3 l
3 l
1.5 l
2 l
~0.4 l
0.125 l
For robot use the primers are aliquoted 10 l per well as follows:
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10 pmol/µl F+R primer stock (20 pmol of each primer pair)
Water
2 l
8 l
The PCR mix is then aliquoted 20l per well:
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Sterile deionised water (MQ)
10x Taq DNA polymerase buffer (1X at final concentration)
2.0 mM dNTP (20% dUTP) (0.20 mM at final concentration)
50 mM MgCl2 (2.5 mM MgCl2 at final concentration)
DNA (17 ng per reaction minimum)
Platinum Taq DNA polymerase (5U/ l stock, i.e. 0.6U per reaction)
PCR program IGF1:
1. 95°C for 10:00
2. 95°C for 0:40
3. 64°C for 0:30 –0.1 per cycle
4. 72°C for 1:00
5. Go to 2 x3
6. 95°C for 0:40
7. 58°C for 0:30
8. 72°C for 1:00
9. Go to 6 x29
10. 72°C 10:00
11. 4°C for ever
~12 l
3 l
3 l
1.5 l
~0.4 l
0.125 l
Purification of PCR product with Millipore Microcon YM-30 Centrifugal Filter
Devices:
1. Pool together all PCR products per individual into a 1.5ml tube and mix thoroughly. A
multichannel pipette that can aspirate up to 50ul will make this stage more efficient.
2. Using pipette, carefully place 500µl of the PCR sample solution to the filter insert
reservoir of the Microcon Centrifugal Filter Tube. Do not allow the pipette tip to touch
the membrane.
3. Place the filter insert into the centrifuge tube and cap the tube securely.
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Spin the device at 12,000 x g (rcf) for 14mins (fixed-angle rotors 36. 45º or swing-bucket
rotor). The columns were not developed for exactly what we are using them for so care
must be taken at this step. After the first spin, discard the flow through.
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Wash the column by spinning a second time at 12,000 x g for 14mins with 500ul
Millipore water. . Discard the respective filtrate containing primer, excessive salt,
nonlabeled dNTPs
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Invert the column (using tweezers), and place it into a new clean tube. Add 30ul of water
to elute the purified PCR product. Spin at 1000 x g for 3mins. Afterwards approx. 15µl
of purified product is eluted.
Fragmentation of the PCR product:
(19 l total volume):
To 15l of PCR product add and mix thoroughly:
 1 l Epicentre UNG (1U)
 1 l USB SAP (1U)
 2 l 10 x Epicentre UNG buffer
Incubate tubes at 37oC for 1.5 hour
Incubate products (for fragmentation of DNA chain) at 95oC for 30 min
Store fragmented PCR products at –20oC
To monitor fragmentation set up parallel electrophoresis in 1-% agarose gel with 2 l product
incubated at 90oC for 10 min and 2 l non-heated product. Fragmented product should be
seen in heated sample lane.
Primer extension reaction (APEX)
Wash the printed DNA Array slide once with 100 mM NaOH and twice with 95oC distilled
water, the second time the water is at 95 oC. Place washed slides on to a thermoplate to warm
up at 58 oC.
Transfer 9l of fragmented PCR product into a 200ul PCR tube.
Add and mix thoroughly terminator nucleotides into a separate tube:
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50M FITC-ddUTP
50M Cy3-ddCTP
50M Cy5-ddGTP
50M Texas Red-ddATP
0.95l
0.95l
0.95l
0.95 l
Add 4ul of reaction buffer
Add 0.875 of dilution buffer
Add 0.125l of Thermosequenase enzyme.
Transfer ~20ul volume of the above mix to the warmed slide and cover with a coverslip.
Incubate slide in dark and humid chamber at 58oC for 25 min.
Wash slide once with 95 oC distilled water
Imaging times used with Genorama 003:
 120 sec. for G (FITC)
 120 sec. for C (Cy3)
 100 sec. for A (Texas Red)
 60 sec. for U (Cy5)