Fluorescein-dUTP
Flu-12-dUTP - Fluorescein-5(6)-carboxamidocaproyl-[5(3-aminoallyl)2'-deoxyuridine-5'-triphosphate)]. Tri(triethylammonium) salt. M.W.= 994 (free acid)
Can be supplied as a dry powder or water solution (1mg/ml).
Flu-12-dUTP, water solution 1mg/ml
| Catalog # | Pack size | Price, Euro | SHOP |
| 507004 | 40 µl | 160,00 | Add to cart |
Datasheet: Flu-12-dUTP
- Absorption and fluorescence emission spectra of fluorescein-labeled dUTP in pH 8.0 buffer
Application:
Fluorescent labelling of DNA by terminal deoxynucleotidyl transferase or DNA-polymerases
Purity:
>96% (HPLC)
Storage:
-20oC
The most popular approach for DNA PCR-labeling with Flu-dUTP or TAMRA-dUTP is based on the usage of dNTPs mixture which contains Flu (TAMRA)-dUTP and all 4 other dNTP in regular concentrations. The molar ratio dUTP/labeled dUTP (or dTTP/labeled dUTP) can vary from 3:1 to 1:1.
The incorporation efficiency depends mainly on the usage of dTTP or dUTP (the incorporation efficiency of dTTP is slightly better than those for dUTP) and on the enzyme used for PCR. Regular Taq DNA polymerase incorporates dUTP (and especially labeled dUTP) less efficient than Taq DNA polymerase with modified active center (Taq-T from Bioron, cat# 117005, 117025).
Using Taq-T instead of regular Taq DNA polymerase one can get more uniform incorporation of dUTP, labeled dUTP and other modified dNTPs into DNA.
In some special applications one may completely substitute dTTP by Flu (TAMRA)-dUTP and to get DNA with all "T" substituted to Flu (TAMRA)-dUTP. Meanwhile, this 100% labeled DNA will be quite different from regular DNA in terms of electrophoresis mobility, hydrophobic properties, denaturation behavior etc. If all these points can be neglected, one can completely substitute dTTP by Flu (TAMRA)-dUTP.
The regular protocol for DNA -labeling with Flu (TAMRA)-dUTP by PCR (ratio of labeled dUTP-nonlabeled:dTTP is 1:2):
| Reagent | Final concentration | Quantity, for 50 µl of reaction mixture |
| Sterile deionized water | - | variable |
| 10x PCR buffer | 1x | 5 µl |
| 10 mM dNTP mix | 0.2 mM of each | 1 µl |
| Flu (TAMRA)-dUTP, 1 mM | 0.1 mM | 5 µl |
| Primer I | 0.1-1 µM | variable |
| Primer II | 0.1-1 µM | variable |
| Taq-T DNA Polymerase | 1.25 u -2.5 u/50 µl | 1.25 u -2.5 u |
| 100 mM MgCl2 | 1-4 mM | variable |
| Template DNA | 10 pg-1 µg | variable |
PCR should be performed as optimized on the regular dNTPs - with the same MgCl2 concentration, with the temperatures and cycles optimized for the particular template and primers.
