Genomic DNA Labeling Protocol

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					                    Genomic DNA Labeling Protocol using Klenow enzyme and N6

1. Add 1.5 ug DNA of the sample to be labeled to an eppendorff tube.

                 Note: For high complexity DNAs (e.g. human genomic DNA), the labeling reaction
                 works more efficiently if the fragment size of the DNA is first reduced, which can be
                 routinely accomplished by restriction enzyme digestion (usually DpnII, though other 4-
                 cutters work as well). After digestion, the DNA should be cleaned up by
                 phenol/chloroform extraction / EtOH precipitation, or by the Qiagen PCR purification kit

2. Add ddH20 to bring the total volume to 21 ul. Then add 20 ul of freshly prepared 2.5X random primer /
reaction buffer mix. Boil 5 min, then place on ice. Spin down briefly, and replace on ice.

                 2.5X random primer / reaction buffer mix:
                 125 mM Tris 6.8
                 12.5 mM MgCl2
                 25 mM 2-mercaptoethanol
                 0.6 ug/ul random hexamers N6

                 i.e. for 20ul buffer, mix 12ul random hexamers [1ug/ul], 2.5ul 1M Tris-HCl pH 6.8;
                 0.25ul 1M MgCl2; 0.04ul 2ME and 5.21ul water

3. On ice, add 5 ul 10X dNTP mix.

                 10X dNTP mix for labeled dCTP:
                 1.2 mM each dATP, dGTP, and dTTP
                 0.6 mM dCTP
                 10 mM Tris 8.0, 1mM EDTA

                 10X dNTP mix for labeled dUTP:
                 1.2 mM each dATP, dCTP, and dGTP
                 0.6 mM dTTP
                 10 mM Tris 8.0, 1mM EDTA

                 i.e. for 50ul dNTP mixes for labeled dCTP, mix 6ul of 10mM dATP, dTTP, dGTP; 3ul of
                 10mM dCTP and 29ul sterile water, can be stored at –20C for a couple of weeks

4. Add 2 ul Cy5-dCTP, or Cy3-dCTP (GE Healthcare cat# PA55321).

                 Note: dCTP and dUTP work equally well.

5. Add 2 ul Klenow enzyme [5U/ul] NEB cat# M0210L.

                 Note: High concentration Klenow (50 units/ul), available through NEB cat# M0210M or
                 Gibco/BRL (as part of the BioPrime labeling kit), supposedly produces better labeling.

6. Incubate 37C overnight, then stop reaction by adding 5 ul 0.5 M EDTA pH8.0

7. Purification of the probes using the QIAquick PCR Purification Kit (Qiagen, cat# 28106). Repeat the
washing step!!!! Elution in 50ul of 0.1X elution buffer [i.e. 1mM Tris pH8.0], or water.
8. Minimize volumes of probes to 20 ul in a speedy vac (Note: it might be better to prevent the probes from
drying out completely and resuspending in water… For some reason, hybs seemed to work slightly better if
the probe had not completely dried out.) If probe volume after speedy vac is less than 20 ul, add sterile

9. Combine two probes (as desired) in one tube, and bring volume up to 40ul with water.

                 Note: adding 30-50 ug human Cot-1 DNA (Gibco/BRL; 1 mg/ml stock; blocks
                 hybridization to repetitive DNAs if present on array), 100 ug yeast tRNA (Gibco/BRL;
                 make a 5 mg/ml stock; blocks non-specific DNA hybridization), and 20 ug poly(dA)-
                 poly(dT) (Sigma catalog No. P9764; make a 5 mg/ml stock; blocks hybridization to
                 polyA tails of cDNA array elements) has been recommended for human arrays

10. Continue with Hybridization protocol