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M. Fero 4/04
adapted from C. O'Neal
Florescent
Labeling Genomic DNA by Random Priming
(for use on BAC microarrays)
Procedure:
Random Prime DNA
- For each sample, combine 300 ng unfragmented genomic DNA and H2O
to bring the volume to 10 µl. (According the manufacturer
as little as 50 ng DNA can be used).
- Add 20 µl of 2.5X Random Primer Mix (Bioprime Kit,
Invitrogen).
- Denature DNA/Primer mix at 100°C for 10min; immediately
quench on ice.
Labeling Reaction
- Combine the following:
30 µl DNA/Random Primer mix
5 µl 10 x dNTP
Mix (see below)
3 µl Cy3-dUTP or Cy5-dUTP
1 µl Klenow (40-50 U/ul)
q.s. 50ul total with H2O
*mix thoroughly and
incubate at 37°C overnight.
Remove
unincorporated nucleotides and add blockering agents
- Combine Cy3 and Cy5 samples.
- Transfer to a G-50 Spin column which has been pre-equillibrated
in TE.
- Spin and collect flow through according to spin column
protocol.
- Add the following directly to the column that contains your
filtered samples:
50 µl Human Cot-1 DNA (1
µg/µl)
10 µl Yeast tRNA (10 µg/ul)
- Dry sample in SpeedVac and store at –20ºC.
Materials
| Description |
Catalog |
Vendor |
Price
|
| BioPrime labeling kit (Klenow) |
18094-011 |
Invitrogen |
214 |
| Yeast tRNA, 25mg |
15401-011 |
Invitrogen |
65 |
| Human Cot-1 DNA, 500 µg/EA |
15279-011 |
Invitrogen |
75
|
| Cy5-dUTP |
PA55022 |
Amersham |
295 |
| Cy3-dUTP |
PA53022 |
Amersham |
29 |
Quick Spin Sephadex G-50 Column,
50 columns/EA
|
1 274 015
|
Roche
|
132
|
Additional Reagents
10 x dNTP Mix: 1.2mM each of dATP, dGTP, dCTP
0.6mM dTTP
10mM Tris pH8.0
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