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One Shot™ Stbl3™ Chemically Competent E. coli
One Shot Stbl3 Chemically Competent E. coli are designed for cloning direct repeats found in lentiviral expression vectors as theseRead more
| Catalog Number | Quantity |
|---|---|
| C737303 | 21 x 50 μL/tube |
Catalog number C737303
Price (USD)
484.00
Special offer
Online exclusive
528.00Save 44.00 (8%)
Each
-
Quantity:
21 x 50 μL/tube
Price (USD)
484.00
Special offer
Online exclusive
528.00Save 44.00 (8%)
Each
One Shot Stbl3 Chemically Competent E. coli are designed for cloning direct repeats found in lentiviral expression vectors as these cells reduce the frequency of homologous recombination of direct repeats found in lentiviral vectors (ViraPower Lentiviral Expression kits) and other retroviral vectors (instability-prone HIV-based plasmids). The Stbl3 strain has been essential for many applications, including genome editing CRISPR workflows, vaccine development (including SARS-CoV-2 vaccine research), cloning, and maintaining vectors with multiple repeats (e.g., gene coding protein with repeated sequence). The transformation efficiency of One Shot Stbl3 chemically competent cells is >1 x 108 cfu/μg plasmid DNA.
The Stbl3 strain is derived from the HB101 E. coli strain with a hybrid K-12 and B strain genome. It contains the recA13 allele of the recA mutation that reduces nonspecific recombination occurrence in cloned DNA. Mutations in the Stbl3 methylation-dependent restriction system (mcrB, mrr, and hsdS20) allow construction of cloning genomic DNA. This strain has endA1+, a nonspecific endonuclease I that digests DNA. The Stbl3 strain also reproducibly yields more plasmid DNA than DH strains (10-fold or more plamsmid DNA than the TOP10 strain) and sustainably produces high titer lentivirus.
One Shot Stbl3 Chemically Competent E. coli offer:
• Expected efficiency of >1 x 108 transformants/μg plasmid DNA
• Optimal performance for the propagation of lentiviral vectors and maintenance of direct repeats
• Enhanced genomic DNA cloning capacity (mcrB, mrr, and hsdS20)
• Capable of producing high DNA yields
Note: For optimal performance and to maintain plasmid with complex repeat sequences, Stbl3 cells should be cultured at 30°C. When performing plasmid DNA isolation from Stbl3 cells that have wild type endonuclease I (endA1+), ensure that buffers contain 10 mM EDTA. EDTA will inactivate the endonuclease and avoid DNA nicking and vector degradation (follow plasmid DNA purification kit instructions for endA1+ E. coli strains).
Easy-to-use One Shot format
Stbl3 Chemically Competent E. coli cells are supplied in the convenient, single-reaction One Shot format. The single-tube, single-use format allows all steps of the transformation protocol, up to plating, to take place in the same tube, thereby helping save time and prevent contamination.
Genotype
F–mcrB mrr hsdS20(rB–, mB–) recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20(StrR) xyl-5 λ–leu mtl-1
Find the strain and format that fit your needs
Other Stbl strains are available with different genetic background.
The Stbl3 strain is available in MultiShot format for high throughput applications.
Explore bacterial growth media formats
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Antibiotic Resistance Bacterial(Yes) Streptomycin
Blue/White ScreeningNo
Cloning Methylated DNAYes (mcrB, mrr)
Cloning Unstable DNAYes (recA13)
Contains F' EpisomeNo
High-throughput CompatibilityLow
Improves Plasmid QualityNo
PlasmidMay be used for plasmids >20 kb
Preparing Unmethylated DNANo
Product LineOne Shot™
Product TypeChemically Competent Cells
Quantity21 x 50 μL/tube
Reduces RecombinationYes (recA13)
Shipping ConditionDry Ice
T1 Phage - Resistant (tonA)No
Transformation Efficiency LevelMedium Efficiency (1 x 108 to 1 x 109 cfu/μg)
FormatTube
SpeciesE. coli (K12/B hybrid)
Unit SizeEach
Contents & Storage
• One Shot Stbl3 Competent E. coli (21 x 50 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 (6 mL)
Store S.O.C. Medium at 4°C or room temperature.
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Store Competent Cells at –80°C.
• pUC19 DNA (50 μL at 10 pg/μL)
Store pUC19 DNA at –20°C.
• S.O.C. Medium (6 mL)
Store S.O.C. Medium at 4°C or room temperature.
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Ready-to-use Bacterial Growth Media
See how these mediums can help with critical aspects of cloning!
Invitrogen One Shot LB Agar* ›
*Only available in North America and selected European countries
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Safety Data Sheets
SDSProduct Information
Frequently asked questions (FAQs)
We would recommend our Stbl3 competent cells, as they have been tested for cloning of unstable lentiviral DNA sequences containing direct repeats.
• Use low passage 293FT cells. Do not use 293FT cells beyond passage 20. Freeze down many aliquots and grow for 2–4 passages prior to transfection.
• Passage cells in complete D-MEM containing G418 (500 µg/mL). Supplement the media with "non-essential" amino acids and sodium pyruvate (0.1 mM MEM Non-Essential amino acids and 1 mM MEM Sodium Pyruvate). Use Gibco FBS (Cat. No. 16000-044).
• Plate cells at a density of 5 x 10e6 per 100 mm dish. Cell density is very important. Make sure that the cells are growing well before re-plating prior to the day of transfection. Avoid overgrowth of 293FT cells when passaging.
• When plating for transfection the next day, do not add G418 to the media.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We do not recommend using mini-prep plasmid DNA for lentivirus production. We recommend preparing lentiviral plasmid DNA using the S.N.A.P. MidiPrep Kit (Cat. No. K1910-01) or PureLink HiPure Plasmid Midiprep Kit (Cat. No. K210004) which contain 10 mM EDTA in the Resuspension Buffer. Since lenti DNA midi-preps also often have low DNA yields, we recommend following specific protocols to increase yield—basically, grow cells slowly, use fewer cells per column, and use 100 mL lenti culture for each DNA midi-prep.
Our ViraPower lentiviral expression system is a 3rd generation system with regard to safety features. Our lentiviral expression vectors are derived from wild type HIV, but nearly all the wild type viral proteins (e.g., Vpr, Vpu, Vif, Nef, Tat) have been removed and the HIV envelope is not used. VSV-G (vesicular stomatitis virus G) envelope protein is used instead. Our ViraPower lentiviral expression system can be used with a 2nd generation lentiviral packaging mix. However, our lentiviral packaging mix would not be compatible with a 2nd generation lentiviral expression vector.
We strongly recommend using Stbl3 E.coli for cloning lentiviral constructs. Stbl3 E.coli contain the recA13 mutation in their genotype that helps to minimize the likelihood of unwanted recombination between the LTRs. After transforming into Stbl3 E.coli, we recommend picking colonies and validating the Lenti DNA from mini-preps, using Afl II and Xho I digests before proceeding to midi-preps. In all of our lentiviral vectors, Afl II sites are present in both 5´ and 3´ LTRs, and an Xho I site is present after the 3´ end of the MCS. Assuming Afl II cuts only in the LTR sites, and there are no Afl II or Xho I sites in the insert, 3 DNA fragments are expected to be generated from the Afl II + Xho I digest. Any unexpected DNA fragments can be assumed to be a result of LTR recombination. Only clones with the expected pattern of DNA fragments should be chosen for the subsequent midi-prep.