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Invitrogen™ MAX Efficiency™ DH10B Competent Cells
Description
Includes
MAX Efficiency DH10B Competent Cells: 5 vials, 200μL each (total of 1mL), pUC19 DNA (0.01μg/mL): 1 vial, 100μL, S.O.C. medium: 2 bottles, 6mL each
MAX Efficiency DH10B Competent Cells are highly efficient E. coli suitable for a variety of molecular biology applications. This strain is one of the most commonly used strains for everyday applications due to key strain features such as high transformation efficiency, making them ideal for generating plasmid libraries, an ability to take up and maintain large plasmids (seen with plasmids of 150 kb in size), and a lack of methylation-dependent restriction systems.
Benefits of MAX Efficiency DH10B Competent Cells
• Ability to maintain large DNA plasmids
• Resistant to the effects of ligase and ligase buffer and tolerant of small amounts of undiluted ligation reactions
• Suitable for the construction of cDNA or genomic DNA libraries using plasmid-derived vectors
Universal strain for everyday applications
The presence of the mcrA genotypic marker and the deletion of mcrBC and mrr makes this strain suitable for cloning DNA that contains methylcytosine and methyladenine. Therefore, genomic DNA, both prokaryotic and eukaryotic, can be cloned efficiently in DH10B.
Versatile DH10B genotype features
• lacZ∆M15 for blue/white screening of recombinant clones
• Elimination of mcrA, mcrBC, mrr, and hsdRMS restriction systems to allow construction of more representative genomic libraries
• endA1 mutation to increase plasmid yield and quantity
Genotype
F–mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara-leu)7697 galU galK λ–rpsL(StrR) nupG
Find the strain and format that fits your need
• DH10B Cells are available in both electrocompetent and chemically competent formats.
• MegaX DH10B T1R Electrocomp Cells (Cat. No.C640003) have the highest transformation efficiency (>3 x 1010 cfu/μg plasmid DNA).
• Multishot formats are available for high throughput applications.
Order Info
Shipping Condition: Dry Ice
Specifications
Specifications
| Product Type | Chemically Competent Cells |
| Contains F' Episome | No |
| Improves Plasmid Quality | Yes (endA1) |
| Cloning Methylated DNA | Yes (mcrA) |
| Transformation Efficiency Level | High Efficiency (>1 x 109 cfu/μg) |
| Content And Storage | • MAX Efficiency DH10B Competent Cells (5 x 200 μL) Store Competent Cells at –70°C • pUC19 DNA (100 μL at 0.01 μg/mL) Store pUC19 DNA at –20°C. • S.O.C. Medium (2 x 6 mL) Store S.O.C. Medium at 4°C or room temperature. |
| Antibiotic Resistance Bacterial | Yes (Streptomycin) |
| Cloning Unstable DNA | Not suitable for cloning unstable DNA |
| Blue/White Screening | Yes (lacZΔM15) |
| High-throughput Compatibility | Low |
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Frequently Asked Questions (FAQs)
For best results, DNA used in electroporation must have a very low ionic strength and a high resistance. A high-salt DNA sample may be purified by either ethanol precipitation or dialysis.
The following suggested protocols are for ligation reactions of 20ul. The volumes may be adjusted to suit the amount being prepared.
Purifying DNA by Precipitation: Add 5 to 10 ug of tRNA to a 20ul ligation reaction. Adjust the solution to 2.5 M in ammonium acetate using a 7.5 M ammonium acetate stock solution. Mix well. Add two volumes of 100 % ethanol. Centrifuge at 12,000 x g for 15 min at 4C. Remove the supernatant with a micropipet. Wash the pellet with 60ul of 70% ethanol. Centrifuge at 12,000 x g for 15 min at room temperature. Remove the supernatant with a micropipet. Air dry the pellet. Resuspend the DNA in 0.5X TE buffer [5 mM Tris-HCl, 0.5 mM EDTA (pH 7.5)] to a concentration of 10 ng/ul of DNA. Use 1 ul per transformation of 20 ul of cell suspension.
Purifying DNA by Microdialysis: Float a Millipore filter, type VS 0.025 um, on a pool of 0.5X TE buffer (or 10% glycerol) in a small plastic container. Place 20ul of the DNA solution as a drop on top of the filter. Incubate at room temperature for several hours. Withdraw the DNA drop from the filter and place it in a polypropylene microcentrifuge tube. Use 1ul of this DNA for each electrotransformation reaction.
There are a few exceptions, but in general the difference is in guaranteed transformation efficiency as follows:
Subcloning Efficiency cells are guaranteed to produce at least 1.0 x 10E6 transformants per µg of transformed pUC19 or pUC18 supercoiled plasmid
Library Efficiency cells are guaranteed to produce at least 1.0 x 10E8 transformants per µg pUC19 or pUC18 DNA
MAX Efficiency cells are guaranteed to produce at least 1.0 x 10E9 transformants per µg pUC19 or pUC18 DNA
Yes. Bacterial host cells will often degrade incoming DNA that has a methylation pattern that is "foreign" relative to that of the cell. Several host strains have been modified to accept mammalian methylation patterns. The modified markers include mcrA, mcrBC, and mrr. Also, endogenous (b-type) restriction endonucleases can be problematic. Modifications of the host to be rK- or rB- are necessary and include hsdR17(AK-, MK+), hsdR17(rK-, mK-), hsdS20(rB-, rB-) or hsdRMS. Strains with the hsdR17(rK-, mK+) mutation lack K-type restriction endonuclease, but contain K-type methylase. DNA prepared from hosts that are rK- mK- is unmethylated and will transform with lower efficiency in rK+ hosts.
TOP10, DH10B, and OmniMAX2-T1 cells contain the mcr, mrr, and hsdRMS mutations. Mach1 and standard DH5? strains only have the hsdR17(rK- mK+) mutation and are not recommended for cloning eukaryotic genomic DNA.
Cosolvents may be used when there is a failure of amplification, either because the template contains stable hairpin-loops or the region of amplification is GC-rich. Keep in mind that all of these cosolvents have the effect of lowering enzyme activity, which will decrease amplification yield. For more information see P Landre et al (1995). The use of co-solvents to enhance amplification by the polymerase chain reaction. In: PCR Strategies, edited by MA Innis, DH Gelfand, JJ Sninsky. Academic Press, San Diego, CA, pp. 3-16.
Additionally, when amplifying very long PCR fragments (greater than 5 kb) the use of cosolvents is often recommended to help compensate for the increased melting temperature of these fragments.
There are several approaches that can be taken to inactivate the AmpliTaq DNA Polymerase after PCR.
(1) Because AmpliTaq DNA Polymerase is thermostable, it is necessary to heat it to high temperatures in order for it to be inactivated. Typically, a 99-100 degrees C for 10 min is sufficient.
(2) Raising the EDTA concentration to 10 mM will chelate any free Mg2+. Mg2+ is necessary for enzyme activity. By removing the Mg2+ the enzyme will no longer exhibit enzyme activity.
(3) Phenol-chloroform extraction of the PCR product and ethanol precipitation will also inactivate AmpliTaq DNA Polymerase.
For Research Use Only. Not for use in diagnostic procedures.