As formulated into most premade reaction kits, application of ung contamination control therefore consists of nothing more than adding the ung to dUTP
containing master mix (if not already present), and adding a few-minute PCR reaction preincubation step at moderate temperatures (usually 37[degrees]-45[degrees]C).
Una aplicacion relevante de implementacion de un sistema de DUTP
en Chile lo constituyen las Areas de Manejo y Explotacion de Recursos Bentonicos (AMERB).
Abbreviations: CFL, compact fluorescent lamp; ERG, electroretinography; H&E, hematoxylin and eosin staining; IHC, immunohistochemistry; ROS, reactive oxygen species; TEM, transmission electron microscopy; TUNEL, terminal deoxynucleotidyl transferase dUTP
nick end labeling.
Terminal deoxynucleotidyl transferase-mediated dUTP
nick end-labeling assay (TUNEL) was performed with a TACS 2 TdT-DAB kit (Trevigen, Gaithersburg, MD) following the manufacturer's instructions.
(10) Several techniques were described to assess the DNA integrity of sperm, and these techniques include TdT (terminal deoxyribonucleotidyl transferase)-mediated dUTP
nick-end labeling (TUNEL) assay, aniline blue straining.
Briefly, 1 [micro]L purified RNA was amplified in a 25-[micro]L reaction containing 2.5 U rTth DNA polymerase (Applied Biosystems), 5 [micro]L of 5 X EZ buffer A, 300 [micro]mol/L of each dNTP (except 600 [micro]mol/L for dUTP
), 3 mmol/L manganese acetate, 0.25 U AmpErase UNG, 0.2 [micro]mol/L forward primer (5'-ATAGATAGCA GGGCAGTTGG-3', corresponding to nucleotides 916-935 of the HA-encoding sequence), 0.2 [micro]mol/L reverse primer (5'-GATCAATTGCCGATTGAGTG-3', complementary to nucleotides 1137-1156 of the HA-encoding sequence), and 0.2 [micro]mol/L probe (5' FAM-CCYTCYCCYTGTGCRTTYTG-BHQ1-3', corresponding to nucleotides 1096-1115 of the HA-encoding sequence).
Next the embryos were incubated in fluorescein-conjugated dUTP
and TdT (TUNEL reagent; In situ Cell Detection kit, Roche Diagnostic, Germany) for 1 h at 38[degrees]C and 5% C[O.sub.2] in the air.
Four [micro]m paraffin sections were used for ISEL with digoxigenin-deoxyuridine (dUTP
) by terminal deoxynucleotidyl transferase (TdT) using an ApopTag peroxidase kit (Appligene Oncor, Illkirch, France).
TUNEL (Terminal deoxynucleotidyl Transferase-mediated dUTP
Nick End Labeling) has been done by mammary tissue were sliced, then drooped with 50ul of TUNNEL labeling mix (consisting of 5ulenzymeterminal deoxynucleotydil transferase and 45ulfluorescein-dUTP), incubated at 37 [degrees]C for 30 minutes in the moist chamber.
PCR was performed in a 50-u.L reaction volume with 1 [micro]L of deoxynucleoside triphosphate mix (12.5 mmol/L each of dATP, dCTP, dGTP, and 5 mmol/L of dUTP
; Applied Biosystems, Foster City, CA, USA), 5 [micro]L of Mg[Cl.sub.2] (25 mmol/L; Applied Biosystems), 0.2 [micro]M each primer, 2.5U of AmpliTaq Gold polymerase (Applied Biosystems), 5 [micro]L of 10 x PCR buffer (Applied Biosystems), and 5 [micro]L of DNA.
Posteriormente, los cortes fueron incubados en una mezcla conteniendo la enzima deoxinucleotidil transferasa terminal (TdT), dUTP
unida a una molecula de deteccion como digoxigenina y dATP en camara humeda a 37 [degrees]C durante una hora y luego se lavo con PBS.