Zero Blunt™ TOPO PCR Cloning Kit for Subcloning

El kit Zero Blunt™ TOPO™ PCR Cloning para subclonación combina la exclusiva tecnología Zero Background™ con el vector pCR™- BluntMás información

Número de catálogo	Cantidad
K283020 también denominado K2830-20	25 reacciones

Número de catálogo K283020

también denominado K2830-20

Precio (MXN)

Cantidad:

25 reacciones

El kit Zero Blunt™ TOPO™ PCR Cloning para subclonación combina la exclusiva tecnología Zero Background™ con el vector pCR™- Blunt II-TOPO™ (Figura 7) para permitir la clonación fácil y de alta eficacia de productos de PCR de extremo romo.
• Tecnología única para minimizar el fondo
• Obtenga su clon: eficiencia del 95 %
• Los múltiples sitios de primer (T7, T3, M13F, M13R) hacen que el análisis de secuencias o PCR sean fáciles y cómodos
• Genes de resistencia a kanamicina y Zeocin en E. coli para elegir
• Sitios de restricción prácticos y validados en MCS para una subclonación adicional
• Incluye células competentes MachI™ T1R para una rápida minipreparación y acorta el tiempo de clonación

Para uso exclusivo en investigación. No apto para uso en procedimientos diagnósticos.

Especificaciones

Cepa bacteriana o de levaduraMach1™

Método de clonaciónBlunt TOPO™

Para utilizar con (aplicación)Biología de la cromatina

Línea de productosOne Shot

Tipo de productoKit de clonación de PCR

Cantidad25 reacciones

VectorVectores de clonación de ADN romos

FormatoKit

Unit SizeEach

Contenido y almacenamiento

Caja 1:
• Vector pCR™Blunt II-TOPO™ linealizado y activado con la topoisomerasa I
• Solución salina
• dNTP
• Plantilla de control
• Primers directos e inversos M13
• Agua estéril

Almacenar entre -5 y -30 °C.
Todos los reactivos son estables durante 6 meses si se almacenan adecuadamente.

Caja 2:
• E. coli químicamente competente One Shot™ o Electrocomp™
• Medio S.O.C.
• Plásmido de control superhelicoidal pUC19

Almacenar a entre -68 y -85 °C.

Preguntas frecuentes

What is the best molar ratio of PCR product:vector to use for TOPO TA cloning? Is there an equation to calculate the quantity to use?

We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1. The quantity used in a TOPO cloning reaction is typically 5-10 ng of a 2 kb PCR product.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 (insert:vector ratio)

What is the best ratio of insert:vector to use for cloning? Is there an equation to calculate this?

The optimal ratio is 1:1 insert to vector. Optimization can be done using a ratio of 0.5-2 molecules of insert for every molecule of the vector.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio

I'm seeing a lot of vector-only colonies when I try to perform a negative control reaction using vector only (no insert) for a TOPO reaction. Is my TOPO vector re-ligating?

Using the vector only for transformation is not a recommended negative control. The process of TOPO-adaptation is not a 100% process, therefore, there will be “vector only” present in your mix, and colonies will be obtained.

I'm trying to clone in my phosphorylated PCR product into a TOPO vector, and I'm getting no colonies. However, when I clone the same product into a TA vector, everything works perfectly. Why is this?

Phosphorylated products can be TA cloned but not TOPO cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. TOPO vectors have a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. Non-TOPO linear vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO cloning.

I'm able to get a lot of colonies, however, none contain my insert of interest. What should I do?

You may be cloning in an artifact. TA and TOPO Cloning are very efficient for small fragments (< 100 bp) present in certain PCR reactions. Gel-purify your PCR product using either a silica-based DNA purification system or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)

View all Zero Blunt™ TOPO PCR Cloning Kit for Subcloning FAQs