La ligasa de ADN T4 cataliza la formación de enlaces fosfodiéster en presencia de ATP entre ADN bicatenario con extremosMás información
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Número de catálogo
Cantidad
46300018
2 x 1 ml
Número de catálogo 46300018
Precio (MXN)
-
Cantidad:
2 x 1 ml
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La ligasa de ADN T4 cataliza la formación de enlaces fosfodiéster en presencia de ATP entre ADN bicatenario con extremos 3´-hidroxilo y 5´-fosfato. El exclusivo tampón de ligasa de ADN T4 optimiza la ligadura, que puede realizarse en 5 minutos (1). Los ácidos nucleicos de una sola cadena no son sustratos para esta enzima. Hay disponible un boletín técnico para ligasa de ADN T4. Aplicaciones: Clonación (ligación con extremo romo o extremo cohesivo) (2). Adición de enlaces o adaptadores a ADN de extremo romo (2).
Fuente: Purificado a partir E. coliœ lisógeno NM989.
Pruebas de rendimiento y calidad: Ensayos de endodesoxirribonucleasa y exdesoxirribonucleasa 3´ y 5´; ligadura de eficacia probada.
Definición de la unidad: Una unidad cataliza el intercambio de pirofosfato marcado como 1 nmol 32P en ATP en 20 min a 37 °C. (Una unidad es igual a aproximadamente 300 unidades de ligadura de extremo cohesivo.)
Condiciones de reacción de la unidad: 66 mM de Tris-HCl (pH 7,6), 6,6 mM de MgCl2, 10 mM de DTT, 66 µM de ATP, pirofosfato marcado como 3,3 µM 32P y enzima en 0,1 ml durante 20 min a 37 °C.
Para uso exclusivo en investigación. No apto para uso en procedimientos diagnósticos.
Especificaciones
Tampón compatibleTampón de reacción 5X
Tipo de productoTampón de ligasa de ADN T4
Cantidad2 x 1 ml
Condiciones de envíoAprobado para su envío en hielo húmedo o seco
Unit SizeEach
Contenido y almacenamiento
ADN ligasa T4 se suministra con un vial de tampón de reacción 5X [250 mm de Tris-HCl (pH de 7,6), 50 mm de MgCl2, 5 mm de ATP, 5 mm de DTT, polietilenglicol-8000 al 25 % (p/v)]. Almacenar a -20 °C.
Preguntas frecuentes
What are common inhibitors of the T4 DNA ligase?
dATP is a competitive inhibitor. Phosphate will reduce ligation efficiency. Detergents in your ligation buffer will likely not affect activity. High levels (0.2M) Na2+, K+, Cs+, Li+, and NH4+ inhibit the enzyme almost completely. Polyamines, spermine, and spermidine also serve as inhibitors.
Do both my insert and vector have to be phosphorylated for successful ligation?
At least one molecule in a ligase reaction (i.e., insert or vector) must be phosphorylated. Ligation reactions are dependent on the presence of a 5' phosphate on the DNA molecules. The ligation of a dephosphorylated vector with an insert generated from a restriction enzyme digest (phosphorylated) is most routinely performed. Although only one strand of the DNA ligates at a junction point, the molecule can form a stable circle, providing that the insert is large enough for hybridization to maintain the molecule in a circular form.
Which T4 DNA Ligase protocol do you recommend when ligating an insert containing one cohesive (sticky) end and one blunt end?
For cloning an insert with one cohesive end and one blunt end, use the conditions for blunt ends. The sticky end may ligate quickly, but the blunt end ligation will still be inefficient. You should use the more stringent protocol to optimize the blunt end ligation. This usually means using more enzyme (5 U), a lower reaction temperature (14C) and a longer incubation time (16-24 hours).
Why is it necessary to dilute ligated DNA products before adding them to competent bacterial cells?
Components of the ligation reaction (enzymes, salts) can interfere with transformation, and may reduce the number of recombinant colonies or plaques. We recommend a five-fold dilution of the ligation mix, and adding not more than 1/10 of the diluted volume to the cells. For best results, the volume added should also not exceed 10% of the volume of the competent cells that you are using.
What are the recommended conditions for blunt-ended ligations?
Generally, ligations are done in a 20 µL volume. Use a total of 100 to 1000 ng of DNA with an insert to vector ratio of 3:1. Add 1.0 units (Weiss) ligase to the reaction. Incubate at room temperature for 4 h or overnight at 14-16 degrees C.
Ideally, assemble several reactions with varying ratios of vector:insert (i.e. 3:1, 5:1, 10:1, 20:1, etc.) to determine the optimal ratio for ligation.
Thermo Fisher Scientific offers T4 DNA ligase at two concentrations: 1 U/µL (Cat. No. 15224-017) and 5 U/µL (Cat. No. 15224-041). When performing blunt or TA cloning ligations, the higher concentration of ligase is generally preferred since ligating a blunt or single base overhang requires more enzyme.