Células competentes MAX Efficiency™ DH10Bac

Citas y referencias (4)

Gibco™

Células competentes MAX Efficiency™ DH10Bac

Las células competentes MAX Efficiency DH10Bac se emplean para la producción de bácmidos recombinantes empleados en el sistema de expresiónMás información

Número de catálogo	Cantidad
10361012	5 x 100 μl

Número de catálogo 10361012

Precio (MXN)

Cantidad:

5 x 100 μl

Las células competentes MAX Efficiency DH10Bac se emplean para la producción de bácmidos recombinantes empleados en el sistema de expresión de baculovirus Bac-to-Bac. La cepa E. coli DH10Bac contiene un vector de transporte del baculovirus (bácmido) que se puede recombinar con plásmido de donante, pFastBac, para crear un bácmido de expresión que contiene un gen de interés clonado. Las características de las células competentes MAX Efficiency DH10Bac incluyen:

• Producción uniforme de > 1 × 10⁸ transformantes/µg
• Tolerancia de pequeñas cantidades de reacciones de ligación sin diluir
• Permite la detección eficaz de colonias azules/blancas (marcador Φ80IacZΔM15)

Uso de células competentes MAX Efficiency DH10Bac
Las células competentes MAX Efficiency DH10Bac pueden servir de huésped para un vector pFastBac recombinante que contiene un gen de interés clonado. Las células DH10Bac presentan un vector de transporte del baculovirus (bMON14272) y un plásmido auxiliar (pMON7142), y son capaces de soportar una recombinación específica del centro entre pFastBac y bMON14272 para generar bácmidos de elevado peso molecular que se podrán amplificar, purificar y emplear para una transfección de células de insectos y su posterior expresión génica. La resistencia a la kanamicina para la selección y el mantenimiento de bácmidos se debe a bMON14272, y la resistencia a la tetraciclina, a pMON7124.

Genotipo:F^-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ^-rpsL nupG/pMON14272/pMON7124

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

Especificaciones

Cepa bacteriana o de levaduraDH10Bac

Tramado azul/blancoSí

Tipo de célulaQuímicamente competente

Clonación de ADN metiladoNo

Clonación de ADN inestableNo es adecuado para clonar ADN inestable

Contiene el episoma F'Carece de episoma F'

Eficacia> 1x10^8

Compatibilidad de alto rendimientoNo compatible con alto rendimiento (manual)

Mejora la calidad de los plásmidosNo

Preparación de ADN no metiladoNo es adecuado para preparar ADN no metilado

Línea de productosDH10Bac, MAX Efficiency

Tipo de productoCélulas competentes

Propagación de vectores ccdBNo para propagación vectorial ccdB

Cantidad5 x 100 μl

Reduce la recombinaciónNo

Condiciones de envíoHielo seco

Resistente al fago T1 (tonA)No

Nivel de eficiencia de transformaciónEficacia media (10^8-10^9 ufc⁄µ g)

FormatoTubo

EspecieE. coli

Unit SizeEach

Contenido y almacenamiento

Contiene:
• Células químicamente competentes MAX Efficiency DH10Bac: 5 viales, 100 µl cada uno (total de 500 µl)
• ADN pUC19 (0,01 µg/ml): 1 vial, 100µ l
•, medio SOC: 2 frascos, 6 ml cada uno

Almacenar las células competentes a -80 °C. Almacene pUC19 DNA a -20 °C. Almacenar el medio S.O.C. a 4 °C o a temperatura ambiente.

Preguntas frecuentes

I cannot detect any recombinant fusion protein after using the BaculoDirect Expression Kit. What could be the cause for this and what do you suggest I try?

Please check the construction of your entry clone, and ensure that the insert is in frame with the vector. Analyze the recombinant viral DNA by PCR to confirm the correct size and orientation of your insert after the LR reaction. Sequence your PCR product to verify the proper reading frame for expression of the epitope tag.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm getting a low-titer P1 viral stock and would like to generate a high-titer stock. What should I do?

To get a high-titer stock, reinfect cells with the P1 stock and generate a P2 high-titer stock. Follow the directions in the BaculoDirect manual on page 18 to generate your P2 stock.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I performed an LR reaction, followed by transfection into Sf9/Sf21 insect cells, but do not see any signs of infection even though it's been 72 hours. What should i do?

Please see our recommendations below:

- Check the LR reaction by PCR analysis prior to transfection into insect cells.
- We recommend using Grace's Insect Cell Culture Medium, Unsupplemented during the transfection experiment instead of serum-free medium, as components in serum-free medium may interfere with transfection.
- Ensure that FBS, supplements, or antibiotics are not included during transfection, as the proteins in these materials can interfere with the Cellfectin II Reagent.
- Use the LR recombination reaction using the pENTR/CAT plasmid as a positive control and Cellfectin II Reagent only (mock transfection) as a negative control.
- Ensure that cells are in the log phase of growth with >95% viability, and the amount of cells are in accordance with the suggestions in the manual.
- Cells may not show signs of viral infection for up to a week depending on transfection efficiency; continue culturing and monitor cells daily for signs of infection.

I see a precipitate in my ganciclovir solution. What can I do?

Warm the ganciclovir solution in a water bath at 37 degrees C for 5-10 min, then vortex for a few minutes. The precipitate should go back into solution.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I am purifying my secreted protein expressed in insect cells with a His-tagged purification column and getting no yield of my protein. Why and what can I do?

Media used to culture insect cells usually have an acidic pH (6.0-6.5) or contain electron-donating groups that can prevent binding of the 6xHis-tagged protein to Ni-NTA. Amino acids such as glutamine, glycine, or histidine are present at significantly higher concentrations in media for growing insect cells than in media for growing mammalian cells, and compete with the 6xHis-tagged protein for binding sites on Ni-NTA matrices. Grace's medium (Thermo Fisher Scientific), for example, contains approximately 10 mM glutamine, 10 mM glycine, and 15 mM histidine.

Dialysis of the medium against a buffer with the appropriate composition and pH (8.0) similar to the lysis buffer recommended for purification under native conditions usually restores optimal binding conditions. Note that depending on the medium used, a white precipitate (probably made up of insoluble salts) can occur, but normally the 6xHis-tagged protein remains in solution. This can be tested by either protein quantitation if using a protein-free medium or by monitoring the amount of 6xHis-tagged protein by western-blot analysis. After centrifugation, 6xHis-tagged protein can be directly purified from the cleared supernatant.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

View all Células competentes MAX Efficiency™ DH10Bac FAQs

Citations & References (4)

Citations & References

Abstract

Molecular characterization of hCTR1, the human copper uptake protein.

Authors: Eisses John F; Kaplan Jack H;

Journal:J Biol Chem

PubMed ID:12034741

'We have expressed hCTR1, the human copper transporter, in Sf9 cells using a baculovirus-mediated expression system, and we observed greatly enhanced copper uptake. Western blots showed that the protein is delivered to the plasma membrane, where it mediates saturable copper uptake with a K(m) of approximately 3.5 microm. We also ... More

Characterization of a heparan sulfate 3-O-sulfotransferase-5, an enzyme synthesizing a tetrasulfated disaccharide.

Authors:Mochizuki H, Yoshida K, Gotoh M, Sugioka S, Kikuchi N, Kwon YD, Tawada A, Maeyama K, Inaba N, Hiruma T, Kimata K, Narimatsu H,

Journal:J Biol Chem

PubMed ID:12740361

Heparan sulfate d-glucosaminyl 3-O-sulfotransferases (3-OSTs) catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 3 of the glucosamine residue of heparan sulfate and heparin. A sixth member of the human 3-OST family, named 3-OST-5, was recently reported (Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J.-P., Malmstrom, ... More

Gene characterized for membrane desaturase that produces (E)-11 isomers of mono- and diunsaturated fatty acids.

Authors: Liu Weitian; Jiao Hongmei; Murray Nancy C; O'Connor Marion; Roelofs Wendell L;

Journal:Proc Natl Acad Sci U S A

PubMed ID:11805319

Moth species have evolved integral membrane desaturases that exhibit a wide diversity in substrate specificity, as well as in regiospecificity and stereospecificity of the unsaturated products. We report here the cloning and expression of a single desaturase from the sex pheromone gland of the light brown apple moth, Epiphyas postvittana, ... More

Reconstitution of ATP-dependent leukotriene C4 transport by Co-expression of both half-molecules of human multidrug resistance protein in insect cells.

Authors: Gao M; Loe D W; Grant C E; Cole S P; Deeley R G;

Journal:J Biol Chem

PubMed ID:8910374

Multidrug resistance protein (MRP) confers a multidrug resistance phenotype similar to that associated with overexpression of P-glycoprotein. Unlike P-glycoprotein, MRP has also been shown to be a primary active ATP-dependent transporter of conjugated organic anions. The mechanism(s) by which MRP transports these compounds and increases resistance to natural product drugs ... More