Invitrogen™ One Shot™ INV110 Chemically Competent E. coli

Catalog No.	Quantity
C717103	21 x 50 μL/tube

Catalog No. C717103 Supplier Invitrogen™ Supplier No. C717103

Description

Includes

One Shot INV110 Chemically Competent E. coli contains 10 transformations of chemically competent cells in One Shot format.

One Shot INV110 Chemically Competent E. coli cells are derived from the JM110 strain and are specifically designed for the growth and purification of plasmid DNA that will be digested with dam or dcm methylation-sensitive restriction enzymes.

One Shot INV110 Chemically Competent E. coli cells are derived from the JM110 strain and are specifically designed for the growth and purification of plasmid DNA that will be digested with dam or dcm methylation-sensitive restriction enzymes. The dam and dcm deficiency allows for the production of plasmid DNA that is unmethylated at GmATC and CmC(A/T)GG sites. INV110 Chemically Competent cells can achieve a transformation efficiency of >1 x 10⁶ cfu/μg of control DNA.

The INV110 strain is a significant improvement over the JM110 strain. The INV110 strain has been engineered to carry an endA1 mutation, which eliminates the non-specific Endonuclease I, for improved plasmid DNA preparation. Mutation in the methylation-dependent restriction system Δ(mcrC-mrr) allows transformation of genomic DNA. This strain also has the lacZΔM15 genotype, providing for the option of blue-white screening on plates containing either X-Gal or Bluo-Gal. The INV110 strain contains an F´ episome that can support cloning plasmids with an f1-like origin of replication (phagemids) and allows isolation of single-stranded DNA (ssDNA). In addition, the F´ episome carries the lacIq repressor for inducible expression from trc, tac, and lac promoters using IPTG.

One Shot INV110 Chemically Competent E. coli offer:
• Transformation efficiencies of >1 x 10⁶ cfu/μg
• dam and dcm deficiency to allow restriction digestion with dam and dcm-sensitive restriction enzymes
• Stable F´ episome that allows isolation of ssDNA and carries lacIq repressor for inducible expression from trc, tac, and lac promoters
• Δ(mcrC-mrr) mutation that reduces cleavage of foreign methylated DNA and improves transformation of genomic DNA
• lacZΔM15 for blue-white color screening of recombinant clones
• endA1 for cleaner DNA preparations and better results in downstream applications due to elimination of nonspecific digestion by Endonuclease I
• Tn10 to permit selection of the host strain using tetracycline

Note: dam deletion results in higher mutation rate, higher recombination frequency, and lower viability on plates. Avoid picking colonies that are noticeably larger than the rest of the population (potential revertants). Pick colonies and isolate the DNA within one day of transformation.

Convenient and efficient format
One Shot INV110 Chemically Competent E. coli cells are supplied in the convenient, single-reaction One Shot format. Each tube contains enough cells for one transformation, reducing both freeze-thaw cycles and costs associated with unused cells.

Genotype
F´ [traD36 proAB lacIqlacZΔM15] rpsL (StrR) thr leu endA thi-1 lacY galK galT ara tonA tsx dam dcm supE44 Δ(lac-proAB) Δ(mcrC-mrr)102::Tn10(TetR)

Find the strain and format that fits your needs 
• We offer other strains in chemically competent and electrocompetent cell formats to meet your specific needs.
• Strains in MultiShot formats for high throughput applications are available.
• Alternative strains for ssDNA production are available, such as TOP10F´ (Cat. No. C303003) or electrocompetent DH12S (Cat. No. 18312017).
• Explore restriction enzyme details to find methylation sensitivity.

Specifications

• Product Type: Chemically Competent Cells
• Contains F' Episome: Yes
• Improves Plasmid Quality: Yes (endA1)
• Cloning Methylated DNA: Yes (mcr, mrr)
• Transformation Efficiency Level: Subcloning Efficiency (1 x 10⁶ to 1 x 10⁷ cfu/μg)
• Content And Storage: • One Shot INV110 Chemically Competent E. coli (21 x 50 μL) • pUC19 vector (20 μL)
  Store at –80°C.
  
  • Recovery Medium
  Store at 4°C or room temperature.
  
  
• Antibiotic Resistance Bacterial: Yes (Streptamycin, Tetracycline)
• Cloning Unstable DNA: No
• Blue/White Screening: Yes (lacZΔM15)
• High-throughput Compatibility: Low
• Preparing Unmethylated DNA: No
• Reduces Recombination: No
• Shipping Condition: Dry Ice
• T1 Phage - Resistant (tonA): Yes
• Species: E. coli (K12)
• Format: Tube
• Product Line: One Shot
• Quantity: 21 x 50 μL/tube

Frequently Asked Questions (FAQs)

I have cloned my gene into my vector and then transformed into TOP10 cells. I then did a plasmid miniprep followed by digestion of the DNA with Xba I. However, the vector is not cutting correctly. Can you help me troubleshoot?

The Xba I cutting site is a Dam-methylation sensitive restriction site. E.coli strains that are dam(+) strains, like TOP10, express the methylating enzyme, Dam. You can try re-transforming into a dam(–) strain, such as INV110. Other dam (and dcm) sensitive restriction sites include the following:
Dam: Bcl I, Cla I, Hph I, Mbo I, Mbo II (Nde II), Taq I, Xba I, BspH I, Nru I
Dcm: Ava II, EcoO 109 I, EcoR II, Sau96 I, ScrF, Stu I, Aat I, Apa I, Ban I, Sfi I

Do you sell a dcm/dam- strain?

Yes, our INV110 strain is dcm/dam- .

What advantages do your Stbl2 cells offer over other cloning strains?

There are other strains available that may function similarly to Stbl2 cells in stabilizing inserts or vectors with repeated DNA sequences. However, one advantage of Stbl2 cells over many similar strains is that they are sensitive to Kanamycin, so you can use Stbl2 to propagate plasmids containing a Kanamycin resistance marker. 

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.

What effects do Dam or Dcm methylase have on restriction enzyme digestion of DNA?

Certain restriction enzymes are unable to recognize and cleave at their target sites if specific adenine or cytosine residues in the sequence are methylated, and Dam and Dcm are two E. coli methylases which introduce methyl groups that affect the cutting sites of many common enzymes. The methylase encoded by the dam gene (Dam methylase) transfers a methyl group from S-adenosylmethionine to the N6 position of the adenine residues in the sequence GATC. The Dcm methylase (encoded by the dcm gene; referred to as the Mec methylase in earlier references) methylates the internal cytosine residues in the sequences CCAGG and CCTGG at the C5 position.

To take advantage of Dam- and Dcm-sensitive restriction enzymes and get proper cleavage, plasmid DNA must be propagated in and isolated from an E. coli strain that is deficient in the endogenous Dam methylase and Dcm methylase enzymes just prior to the restriction reaction. We have one competent cell product available that is made with a dam- and dcm- strain: One Shot INV110 Chemically Competent E. coli (Cat. No. C7171-03).

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.

Why is it necessary to dilute ligated DNA products before adding them to competent bacterial cells?

Components of the ligation reaction (enzymes, salts) can interfere with transformation, and may reduce the number of recombinant colonies or plaques. We recommend a five-fold dilution of the ligation mix, and adding not more than 1/10 of the diluted volume to the cells. For best results, the volume added should also not exceed 10% of the volume of the competent cells that you are using.

When should DMSO, formamide, glycerol and other cosolvents be used in PCR?

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.

For Research Use Only. Not for use in diagnostic procedures.