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ElectroMAX™ Stbl4™ Competent Cells
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ElectroMAX™ Stbl4™ Competent Cells
Invitrogen™

ElectroMAX™ Stbl4™ Competent Cells

ElectroMAX Stbl4 Competent Cells are specifically designed for cloning unstable inserts. This strain is a derivative of Stbl2 cells (engineeredRead more
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116350185 x 100 μL
Catalog number 11635018
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415.00
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5 x 100 μL
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ElectroMAX Stbl4 Competent Cells are specifically designed for cloning unstable inserts. This strain is a derivative of Stbl2 cells (engineered from JM109/J5 strain). To improve performance, the gal- mutation was introduced, lon- restored to wild type lon+ allele, and F′ episome introduced. The Stbl4 strain retains the genetic properties of the Stbl2 strain for the maintenance of unstable inserts such as retroviral sequences, direct repeats, and tandem array genes. In addition, the Stbl4 strain is reportedly used in discovery of complex secondary metabolites and CRISPR library construction and maintenance.

As electrocompetent cells, Stbl4 cells can achieve high transformation efficiency of >5 x 109 cfu/μg, making them ideal for challenging applications such as generating cDNA, CRISPR, and genomic libraries of unstable inserts or using low DNA input. Electrocompetent Stbl4 cells are able to take up and maintain large plasmids (e.g., 50 kb cosmids and 100–200 kb P1 clones). Stbl4 cells also contain an F' episome, allowing them to serve as a host for single-stranded DNA such as M13mp cloning vectors if a lawn of DH5αF′IQ is provided to allow plaque formation. The lacZΔM15 marker provides α-complementation of the β-galactosidase gene from pUC or similar vectors and can therefore be used for blue-white screening of colonies on agar plates containing X-gal or Bluo-gal and IPTG. The mcrA mutation and the mcrBC-hsdRMS-mrr deletion allow cloning of genomic sequences that are methylated. Finally, the endA1 mutation greatly increases plasmid yield and quality.

ElectroMAX Stbl4 competent cell features
• Provide transformation efficiencies of >5 x 109 cfu/μg
• Unique genotype stabilizes direct repeats and retroviral sequences
• The mcrA mutation and mcrBC-hsdRMS-mrr deletion allows cloning methylated genomic sequences
• Support of blue-white screening of recombinant clones due to lacZΔM15
• Designed to deliver high-yield plasmid preparations for downstream applications
• Stable F' episome allows isolation of ssDNA

Propagating unstable and large DNA with high transformation-efficiency cells
Many competent cell strains have the recA1 genotype, which reduces recombination. However, there are some instances when the DNA that you are trying to clone is still unstable in such cells, perhaps due to the presence of inverted or direct repeats or GC-rich tracts. While such sequences are relatively common in eukaryotic genomes, they are rare in E. coli. Consequently, rearrangements may occur when these sequences are introduced into standard E. coli strains.

Genotype: mcrA Δ(mcrBC-hsdRMS-mrr) recA1 endA1 gyrA96 galthi-1 supE44 λ–relA1 Δ(lac-proAB) / F′ [proAB+lacIqZΔM15 Tn10(TetR)]

Find the strain and format that fits your needs
Stbl strains are available with a variety of genetic background.
The Stbl3 strain is available in MultiShot format for high throughput applications.
MAX Efficiency DH5αF´IQ Competent Cells are available in chemically competent format.
Explore bacterial growth media formats

Notes
• To maximize stabilization of direct repeats and retroviral sequences, incubate Stbl4 cells and perform expression studies at 30°C.
• A high-voltage electroporation apparatus is required.
• Stbl4 strain genome sequences are available in the NCBI GenBank database under accession numbers CP076043.1 and CP076044.1 (Assad et al. 2021).

For Research Use Only. Not for use in diagnostic procedures.
Specifications
Antibiotic Resistance BacterialYes (Tetracycline)
Blue/White ScreeningYes (lacZΔM15)
Cloning Methylated DNAYes (mcrB, mrr)
Cloning Unstable DNAYes (recA1)
Contains F' EpisomeYes
High-throughput CompatibilityLow
Improves Plasmid QualityYes (endA1)
PlasmidMay be used for plasmids >100 kb
Preparing Unmethylated DNANo
Product LineElectroMAX™
Product TypeElectrocompetent Cells
Quantity5 x 100 μL
Reduces RecombinationYes (recA1)
Shipping ConditionDry Ice
T1 Phage - Resistant (tonA)No
Transformation Efficiency LevelHigh Efficiency (>1 x 109 cfu/μg)
FormatTube
SpeciesE. coli (K12)
Unit SizeEach
Contents & Storage
• ElectroMAX Stbl4 Competent Cells (5 x 100 μL)
Store Competent Cells at –80°C.

• pUC19 DNA (50 μL at 10 pg/μL)
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.

Ready-to-use Bacterial Growth Media

Ready-to-use Bacterial Growth Media

See how these mediums can help with critical aspects of cloning!

Gibco LB Broth

Invitrogen S.O.C. Medium

Invitrogen One Shot LB Agar*

*Only available in North America and selected European countries

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Frequently asked questions (FAQs)

How do you recommend that I prepare my DNA for successful electroporation of E. coli?

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.

How can I clone a gene that has direct repeats and propagate it without altering the repeat sequences?

The first thing you can do is to lower the growth temperature of your E. coli cells when propagating your plasmid containing the unstable gene. Slowing the growth of any cell strain at 30C, 25C or even lower can help to stabilize the replication of the plasmids they contain.

If your sequence is still unstable despite low-temperature growth, there are also specific bacterial strains available that can further help to stabilize repeated sequences during propagation. Invitrogen Stbl2 and Stbl4 competent cells are both designed to improve stability when cloning retroviral or direct repeat sequences.

In a series of experiments, Stbl2 was compared directly to several other strains also known for increasing stability of retroviral and tandem repeat inserts. An article in the Focus Journal (Issue 16.3, p. 78) contains data from two such experiments – the full article can be found on the Thermo Fisher Scientific website. A brief summary of the data is included below:

Stability of clones containing SIV retroviral sequences:
Stbl2 @ 30°C - 100%; Stbl2 @ 37°C - 100%; HB101 @ 30°C - 100%; HB101 @ 37°C - 100%; SURE @ 30°C - 72%; SURE @ 37°C - 0%

Stability of clones containing 100 repeats of a 32-bp sequence:
Stbl2 @ 30°C - 89%; Stbl2 @ 37°C - 73%; HB101 @ 30°C - 15%; HB101 @ 37°C - 0%; SURE @ 30°C - 53%; SURE @ 37°C - 0%

Results from a separate experiment on stability of a tandem repeat of four R67 dihydrofolate reductase genes in Stbl2 vs. SURE cells can be found in Focus 19.2, p. 24 on the Thermo Fisher Scientific website.

Can encapsulated phagemid DNA or M13 phage be used to infect bacteria?

Single-stranded DNA viral particles like M13 require the presence of an F pilus in order to infect E. coli. This criterion is met by TOP10F', DH5? F'IQ, INV?F', Stbl4, OmniMAX2-T1 and DH12S cells. These cells are not traD mutants, which effectively allows the cells to retain the F' episome. Transforming single-stranded DNA can cause a 100- to 1,000-fold reduction in efficiency compared to viral particles.

Is S.O.C. medium absolutely required when recovering competent bacterial cells during transformation?

Many media can be used to grow transformed cells, including standard LB, SOB or TB broths. However, S.O.C. is the optimal choice for recovery of the cells before plating. The nutrient-rich formula with added glucose is often important for obtaining maximum transformation efficiencies.

How can unstable or toxic DNA inserts be maintained in bacteria?

There are a few steps you can take to improve stability of clones with difficult-to-maintain inserts. Supplement the medium with extra nutrients (e.g., add 20-30 mM glucose to Terrific Broth) or try a vector that has a reduced copy number (e.g., pBR322). Some clones can exhibit a high degree of deletions; this is usually a result of the clones having long terminal repeat (LTR) sequences or regions with high secondary structure. To overcome this problem, the cells can be grown at 30°C or ambient temperature (in LB or in a nutrient rich broth like Terrific Broth). Do not to let the cells reach late stationary phase in liquid culture. Alternatively, transform into cells that maintain unstable sequences such as Stbl2, Stbl3, or Stbl4 cells.

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3156506Certificate of AnalysisMay 07, 202511635018
3088990Certificate of AnalysisFeb 04, 202511635018
3033105Certificate of AnalysisOct 29, 202411635018
3009442Certificate of AnalysisAug 27, 202411635018
2968651Certificate of AnalysisJul 17, 202411635018
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