西亚试剂：：Nick Translation of DNA for CGH

 Prepare reaction mixtures per 50ul Add Enzymes last, gently vortex mixture, quick spin liquid to bottom of tube:

 

 

Label	Biotin-dUTP	Digox-dUTP	FITC-dUTP	Texas Red dUTP
10X Biotin dNTP	5 ul	0 ul	0 ul	0 ul
10X A4 dNTP	0 ul	5 ul	5 ul	5 ul
dUTP	0 ul	1 ul	1 ul	1 ul
DNA POL-1	1 ul	1 ul	1 ul	1 ul
DNA Pol/DNAse (additional)	(2.5-5 ul)	(2.5-5 ul)	(2.5-5 ul)	(2.5-5 ul)
DNA (1ug)
ddH2O
Total Reaction Volume	50 ul	50 ul	50 ul	50 ul

2. Incubate reaction mixtures for 60 minutes* at 15 C (prepare in advance using ice bucket, water and ice).

3. Stop reaction by heating at 70 C for 15 minutes.

4. Run 3-5 ul* of probe on 1% agarose gel to check size. Product should run as a smear ranging from 0.3-2.3 kb.

5. Store probes at -20 C.

NOTES:

The volume of enzyme, time of incubation, and amount to run on agarose gel will vary depending on sample and enzyme:

 

Sample	DNA	Enzyme	Enzyme Vol	Time	Gel Vol	Probe size
Fresh:	1ug	10X	3-5 ul	60-70 min	5 ul (12 comb)	300-2.3 kb (size is critical)
Paraffin	1-2ug	10X	5 ul	70-90 min	5 ul (12 comb)	300-4 kb
PCR Amplified Fresh DNA	25 ul	mix	3.5 ul	60 min	3 ul (16 comb)	100-2 kb
PCR Amplified Microdissected DNA	40 ul	mix	3.0 ul	60 min	3 ul (16 comb)	100-600 bp

The 10X enzyme is the "fast enzyme" and we order the same lot of this for several months. The slow enzyme is a separate enzyme, which requires testing on fresh PCR amplified DNA with FITC and or Texas Red to determine what mixture of fast and slow to use for subsequent nick translations.

SOLUTIONS

10X A4:

• 5 ul of 10 mM dATP, 5 ul of 10 mM dCTP, 5 ul of 10 mM dGTP (Gibco; final is 200uM, 10X)
• 125 ul 1M Tris, pH 7.2 (final 500uM, 10X)
• 12.5 ul 1M MgCl2 (final 200uM, 10X)
• 1.7 ul 14.7 M mercaptoethanol (100mM, 10X)
• 0.5 ul 50 mg/ml BSA (Gibco; final 100 ug/ml, 10X)
• 95.3 ul dH20 to final volume of 250ul.
• Store at -20 C in screw cap tubes.

 

Texas Red-dUTP and FITC-dUTP:

• 25nm from Dupont/NEN (catalog # NEL413 & NEL417; Remember to spin down after thawing, before opening!!!).

Dig-11-dUTP

• 25 nm (Boehringer Mannheim); spin down before opening.

 

DNA Pol-1:

• (Gibco/BRL cat #18010-025)

DNA Polymerase/DNase I:

The 10X Enzyme mix is from the BIONICK kit (GIBCO). If this enzyme is cutting the DNA too small then try varying the amount and/or time of incubation. The standard concentration for fresh DNA is 3 ul of the 10X Enzyme mix for 60 minutes. Incorporation decreases appreciably below 2 ul or less than 40 mins. If the mix is still cutting too small, with these minimum amounts, then try the "slow" enzyme mix (below), or a mixture of the two. We attempt to freeze down one lot of 10X Enzyme with known characterstics to be used for several months.

DNA Pol-1/DNase I: this is the "slow enzyme" mix in the Nick Tranlsation Kit from GIBCO. You can also order the enzyme separately from Gibco (catalog #18162-016). This enzyme is less active than the 10X Enzyme mix from the BioNick kit. If you need to use this then try 60 minutes using 5 ul first, and adjust conditions as needed. This enzyme is better for smaller DNA and/or degraded DNA .

FRESH DNA: This should be of high molecular weight. If the DNA has some degraded lower molecular weight DNA present then the enzyme used for the Nick Translation should probably be the "slow" enzyme as discussed above.

PARAFFIN DNA: Formalin fixed dna appears to be resistant to cutting and incorporation. It is best to use the full 5 ul of 10X fast enzyme for 90 minutes, unless the DNA is getting cut up easily. If the volume of the DNA in TE is greater then 10 ul, add more enzyme to compensate for the extra EDTA from the TE in the reaction

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