One Shot™ INV110 Chemically Competent E. coli - 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.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.