16 Comparative Genomic Hybridization
203
12. Run 5 1..11 of sample on a 1-1.5% agarose gel. Samples amplified from
fixed tissues are routinely smeared below 1000 bp. Unfixed DNA products usually range from 400-1500 bp.
Direct labeling by nick translation for CGH
- lOx A4: 5 ~11O mM dATP, 5 ~11O mM dCTP, 5 ~11O mM dGTP, (Gibco: Reagents
final, 200 ~M), 125 ~IIM Tris, pH 7.2 (finaI500~M), 12.5 ~l 1M MgCl z
(final 200 ~M), 1.7 ~114.7 M mercaptoethanol (final 100 mM), 0.5 ~150
mg/ml BSA (final 100 ~g/ml), 95.3 ~l ddH20 to final volume of250 ~l.
Store at -20°e.
- Fluorescent dUTPs: These are available from a number of vendors. For
example, Texas Red and FITC are available from DuPont/NEN and
Dig-ll-dUTP is from Roche. These should be spun down after thawing
and before opening.
- DNAPollDNAase I: This is the lOx enzyme mix from the Gibco Bionick kit (Life Technologies). If this enzyme cuts DNA too vigorously,
then vary the amount of enzyme or the incubation time. Standard for
fresh DNA is 3 ~l of the lOx mix for 60 min. Incorporation decreases
significantly at less than 2 ~l or 40 min incubation. Ifthe mixture is too
active with these minimum amounts try the "slow" mix from the Gibco
Nick Translation Kit (Life Technologies) or a mixture of the two
("slow" enzyme can also be ordered separately). Try using 5 ~l for
60 min first and adjust conditions as needed. This "slow" enzyme
is best for degraded or low molecular weight DNA.
- Fresh DNA: This should be high molecular weight.
- Paraffin DNA: Formalin-fixed DNA resists enzymatic cutting and nucleotide incorporation. We start by using the fullS ~l of fast enzyme for
90 min unless the DNA is easily cut. If DNA volume in Tris/EDTA is
greater than 10 ~l, more enzyme will be needed to compensate for the
extra EDTA in the reaction.
1. Prepare reaction mixtures in a total of 50 ~l. We use approximately 1~g labeling
fresh DNA and 1 ~l of fluorochrome. Add enzymes last, gently vortex,
and quickly spin down.
2. Incubate reaction mixtures 60 min at IS-16°C for 60 min.
3. Stop reaction by heating to 70-73°C for 15 min.
203
12. Run 5 1..11 of sample on a 1-1.5% agarose gel. Samples amplified from
fixed tissues are routinely smeared below 1000 bp. Unfixed DNA products usually range from 400-1500 bp.
Direct labeling by nick translation for CGH
- lOx A4: 5 ~11O mM dATP, 5 ~11O mM dCTP, 5 ~11O mM dGTP, (Gibco: Reagents
final, 200 ~M), 125 ~IIM Tris, pH 7.2 (finaI500~M), 12.5 ~l 1M MgCl z
(final 200 ~M), 1.7 ~114.7 M mercaptoethanol (final 100 mM), 0.5 ~150
mg/ml BSA (final 100 ~g/ml), 95.3 ~l ddH20 to final volume of250 ~l.
Store at -20°e.
- Fluorescent dUTPs: These are available from a number of vendors. For
example, Texas Red and FITC are available from DuPont/NEN and
Dig-ll-dUTP is from Roche. These should be spun down after thawing
and before opening.
- DNAPollDNAase I: This is the lOx enzyme mix from the Gibco Bionick kit (Life Technologies). If this enzyme cuts DNA too vigorously,
then vary the amount of enzyme or the incubation time. Standard for
fresh DNA is 3 ~l of the lOx mix for 60 min. Incorporation decreases
significantly at less than 2 ~l or 40 min incubation. Ifthe mixture is too
active with these minimum amounts try the "slow" mix from the Gibco
Nick Translation Kit (Life Technologies) or a mixture of the two
("slow" enzyme can also be ordered separately). Try using 5 ~l for
60 min first and adjust conditions as needed. This "slow" enzyme
is best for degraded or low molecular weight DNA.
- Fresh DNA: This should be high molecular weight.
- Paraffin DNA: Formalin-fixed DNA resists enzymatic cutting and nucleotide incorporation. We start by using the fullS ~l of fast enzyme for
90 min unless the DNA is easily cut. If DNA volume in Tris/EDTA is
greater than 10 ~l, more enzyme will be needed to compensate for the
extra EDTA in the reaction.
1. Prepare reaction mixtures in a total of 50 ~l. We use approximately 1~g labeling
fresh DNA and 1 ~l of fluorochrome. Add enzymes last, gently vortex,
and quickly spin down.
2. Incubate reaction mixtures 60 min at IS-16°C for 60 min.
3. Stop reaction by heating to 70-73°C for 15 min.