Nick Translation of DNA for CGH
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Nick Translation of DNA for CGH
| Label | ||||
| 10X Biotin dNTP | ||||
| 10X A4 dNTP | ||||
| dUTP | ||||
| DNA POL-1 | ||||
|
DNA Pol/DNAse (additional) |
||||
| DNA (1ug) | ||||
| ddH2O | ||||
| Total Reaction Volume |
3. Stop reaction by heating at 70 C for 15 minutes.
NOTES:
| Sample | ||||||
| Fresh: | ||||||
| Paraffin | ||||||
| PCR Amplified Fresh DNA | ||||||
| PCR Amplified Microdissected DNA |
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-dU TP:
Dig-11-dUTP
DNA Pol- 1:
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 DN A: 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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