Ligasa de ADN T4 (1 U/μl)
Ligasa de ADN T4 (1 U/μl)
Citas y referencias (3)
Invitrogen™

Ligasa de ADN T4 (1 U/μl)

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 catálogoCantidad
15224025500 U
15224017100 U
152240904 × 500 U
Número de catálogo 15224025
Precio (MXN)
-
Cantidad:
500 U
Pedido a granel o personalizado
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. Los ácidos nucleicos de una sola cadena no son sustratos para esta enzima.

Aplicaciones
Clonación (ligadura con extremo romo o extremo cohesivo) y adición de enlaces o adaptadores a ADN de extremo romo


Purificada a partir del lisógeno lambda de E. coli NM989

Pruebas de rendimiento y calidad
Endodesoxirribonucleasa, ensayos de endodesoxirribonucleasa 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 unitarias
66 mM de Tris-HCl (pH 7,6), 6,6 mM de MgCl2, 10 mM de DTT, 66 µM de ATP, 3,3 µM de pirofosfato marcado como 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 productoT4 DNA Ligase
Cantidad500 U
Condiciones de envíoHielo seco
Concentración1 U/μl
EnzimaLigasa
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 is the difference between T4 DNA Ligase and E.coli DNA Ligase?

The main difference between the 2 enzymes is that E. coli DNA Ligase cannot ligate blunt dsDNA fragments. Both ligases can be used to repair single stranded nicks in duplex DNA and to perform cohesive or sticky end ligations. E. coli DNA Ligase is generally used to seal nicks during second strand cDNA synthesis, since T4 DNA Ligase could result in formation of chimeric inserts.

How can I optimize my ligation reaction?

Please consider the following suggestions:
1– Try different molar ratios of insert to vector. Having an excess of insert is usually what will work, try 1:1 to 15:1 insert:vector.
2– Try increasing the time of the ligation at 37 degrees C.
3– Try performing the ligation at 16 degrees C overnight (you can set it up on your PCR machine).

I cannot transform my cells right away. Can I store my ligation reaction? If so, at what temperature should I store it?

Make sure you have inactivated the ligase and store the ligation reaction at 4 degrees C.

What kind of controls should I have for restriction cloning?

You can have all of the below controls or select the one you consider the most appropriate to the problem you are facing:
1– Transform the E. coli with circular plasmid to assess the competency of the cells (how well they are taking up DNA).
2– Transform and plate the dephosphorylated vector. It will help you assess how well the dephosphorylation worked and what proportion of colonies in your ligation transformation plate could be false positives (re-ligated vector or background).
3– Use T4 DNA igase to re-ligate your cut vector, or lambda DNA/Hind III marker. It will help you assess whether the ligase itself is working properly.

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.

View all Ligasa de ADN T4 (1 U/μl) FAQs

Citations & References (3)

Citations & References
Abstract
Identification of novel non-phosphorylated ligands, which bind selectively to the SH2 domain of Grb7.
Authors: Pero Stephanie C; Oligino Lyn; Daly Roger J; Soden Amy L; Liu Chen; Roller Peter P; Li Peng; Krag David N;
Journal:J Biol Chem
PubMed ID:11809769
'Grb7 is an adapter-type signaling protein, which is recruited via its SH2 domain to a variety of receptor tyrosine kinases (RTKs), including ErbB2 and ErbB3. It is overexpressed in breast, esophageal, and gastric cancers, and may contribute to the invasive potential of cancer cells. Molecular interactions involving Grb7 therefore provide ... More
Presteady-state Analysis of Avian Sarcoma Virus Integrase. I. A SPLICING ACTIVITY AND STRUCTURE-FUNCTION IMPLICATIONS FOR COGNATE SITE RECOGNITION.
Authors: Bao Kogan K; Skalka Anna Marie; Wong Isaac;
Journal:J Biol Chem
PubMed ID:11821409
'Integrase catalyzes insertion of a retroviral genome into the host chromosome. After reverse transcription, integrase binds specifically to the ends of the duplex retroviral DNA, endonucleolytically cleaves two nucleotides from each 3''-end (the processing activity), and inserts these ends into the host DNA (the joining activity) in a concerted manner. ... More
Binding of low affinity N-formyl peptide receptors to G protein. Characterization of a novel inactive receptor intermediate.
Authors: Prossnitz E R; Schreiber R E; Bokoch G M; Ye R D;
Journal:J Biol Chem
PubMed ID:7738006
G protein-coupled seven-transmembrane-containing receptors, such as the N-formyl peptide receptor (FPR) of neutrophils, likely undergo a conformational change upon binding of ligand, which enables the receptor to transmit a signal to G proteins. We have examined the functional significance of numerous conserved charged amino acid residues proposed to be located ... More