Initiation mix (10X; IM)
MgCl2
Nucleotides, phosphorylated
Nucleotides, phosphorylated, modified (optional; see below)
Physiological buffer (PB)
-
1. Add the following concentrations of each of the phosphorylated precursors to PB:
Depending on the labeling and/or detection system to be used for a specific application, add the appropriate modified precursor
in place of the equivalent unmodified precursor.
| Precursor |
Modified precursora (sourceb)
|
Concentration |
| dATP |
Biotin-7-dATP (Enzo Life Sciences) |
250 μM |
| dCTP |
Biotin-11-dCTP (Enzo Life Sciences) |
250 μM |
| dGTP |
|
250 μM |
| dTTP analog |
Biotin-11-dUTP (Enzo Life Sciences; PerkinElmer) |
10-100 μM |
|
Biotin-16-dUTP (Enzo Life Sciences; PerkinElmer) |
|
|
Digoxigenin-11-dUTPc (Roche)
|
|
|
Fluorescein-12-dUTP (Roche; PerkinElmer) |
|
|
Texas Red-5-dUTP (PerkinElmer) |
|
| CTP |
|
100 μM |
| GTP |
|
100 μM |
| UTP |
|
100 μM |
| aModified precursors added to the medium instead of the equivalent unmodified precursor support replication by endogenous eukaryotic
DNA polymerases, but at a reduced rate; elongation is typically 5%-20% of the normal level.
|
| bClosely related compounds not listed above must be tested but can be assumed to be acceptable substrates.
|
| cUse care when handling digoxigenin-labeled compounds.
|
-
2. Add MgCl2 to a molarity equal to the total of the triphosphates.
