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The most popular approach for DNA PCR-labeling with Flu-12-dUTP or TAMRA-dUTP is based on the usage of dNTPs mixture which contains Flu-12-dUTP and all the other 4 dNTs in regular concentrations. The molar ratio of 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 HotScriptase (M3056) from Genaxxon.
In some special applications one may completely substitute dTTP by TAMRA-dUTP to get DNA with all 'T' substituted to TAMRA-dUTP. Meanwhile, this 100% labeled DNA will be quite different from regular DNA in terms of electrophoresis mobility, hydrophobic properties, denaturation behavior etc. it will be no problem to do this just from the technical point of view.
Flu-12-dUTP is supplied as an aqueous solution (1mM).
Purity: >96% (HPLC)
MW = 994 g/mol (free acid)
Regular protocol for labelling of DNA with Flu-12-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||5µL|
|10 mM dNTP Mix||0.2mM each||1µL|
|Primer I||0.1 - 1µM||variable|
|Primer II||0.1 - 1µM||variable|
|HotScriptase||1.25 - 2.5 units/50µL||1.25 - 2.5 units|
|100mM MgCl2||1 - 4 mM||variable|
|Template DNA||10pg - 1µg||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.
application:Fluorescent labelling of DNA by terminal deoxynucleotidyl transferase or DNA-polymerases.
Sicherheits Hinweise / Safety
Klassifizierungen / Classificationeclass-Nr: 34-16-08-06
Dokumente - Protokolle - Downloads
Here you will find information and further literature on Flu-12-dUTP, min. 96% (1mM). For further documents (certificates with additional lot numbers, safety data sheets in other languages, further product information) please contact Genaxxon biosience at: firstname.lastname@example.org or phone: +49 731 3608 123.