MAX Efficiency™ DH10Bac Competent Cells
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MAX Efficiency™ DH10Bac Competent Cells
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MAX Efficiency™ DH10Bac Competent Cells

MAX Efficiency DH10Bac Competent Cells are used for production of recombinant bacmids used in the Bac-to-Bac Baculovirus Expression System. TheRead more
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Catalog number 10361012
Price (USD)
710.00
Each
Add to cart
Price (USD)
710.00
Each
Add to cart
MAX Efficiency DH10Bac Competent Cells are used for production of recombinant bacmids used in the Bac-to-Bac Baculovirus Expression System. The DH10Bac E. coli strain contains a baculovirus shuttle vector (bacmid) that can recombine with a donor plasmid, pFastBac, to create an expression bacmid containing a cloned gene of interest. Features of MAX Efficiency DH10Bac Competent Cells include:

• Consistent yields of >1 × 108 transformants/μg
• Tolerance of small amounts of undiluted ligation reactions
• Support of efficient blue/white colony screening (Φ80lacZΔM15 marker)

Using MAX Efficiency DH10Bac competent cells
MAX Efficiency DH10Bac Competent Cells can serve as a host for a recombinant pFastBac vector containing a cloned gene of interest. DH10Bac cells harbor a baculovirus shuttle vector (bMON14272) and a helper plasmid (pMON7142), and are capable of supporting site-specific recombination between pFastBac and bMON14272 to generate high molecular weight bacmids that can then be amplified, purified, and used for insect cell transfection and subsequent gene expression. Kanamycin resistance for bacmid selection and maintenance is conferred by bMON14272, and tetracycline resistance by pMON7124.

Genotype:F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ-rpsL nupG/pMON14272/pMON7124
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Product TypeCompetent Cells
Contains F' EpisomeLacks F' Episome
Efficiency> 1x10^8
Cell TypeChemically Competent
Improves Plasmid QualityNo
Cloning Methylated DNANo
Transformation Efficiency LevelMedium Efficiency (10^8-10^9 cfu⁄μg)
Cloning Unstable DNANot Suitable for cloning unstable DNA
Bacterial or Yeast StrainDH10Bac
Blue/White ScreeningYes
High-throughput CompatibilityNot High-throughput Compatible (Manual)
Preparing Unmethylated DNANot Suitable for preparing unmethylated DNA
Propagating ccdB VectorsNot for ccdB vector propagation
Reduces RecombinationNo
Shipping ConditionDry Ice
T1 Phage - Resistant (tonA)No
SpeciesE. coli
FormatTube
Product LineDH10Bac, MAX Efficiency™
Quantity5 x 100 μL
Unit SizeEach
Contents & Storage
Contains:
• MAX Efficiency DH10Bac Chemically Competent Cells: 5 vials, 100 μl each (total of 500 μl)
• pUC19 DNA (0.01 μg/ml): 1 vial, 100 μl
• SOC Medium: 2 bottles, 6 ml each

Store Competent Cells at -80°C. Store pUC19 DNA at -20°C. Store SOC Medium at 4°C or at room temperature.

Frequently asked questions (FAQs)

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.

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