Cellules E. coli One Shot™ OmniMAX™ 2 T1^R chimiquement compétentes

OmniMAX 2 is the preferred strain for transforming libraries because of its high transformation efficiency and genomic cloning compatibility characteristics.

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. 

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.

Yes, several of our competent cells products are frozen with DMSO. The presence of DMSO (dimethylsulfoxide) will generally be indicated in the MSDS files if you have a question about a particular product, but here is a list of commonly used products that are known to have DMSO in the freezing buffer:

One Shot OmniMAX 2 T1 Phage Resistant Cells, Cat. No. C8540-03

One Shot INV?F' Chemically Competent Cells, Cat. No. C2020-03 and C2020-06

One Shot MAX Efficiency DH5?-T1 Chemically Competent Cells, Cat. No. 12297-016

MAX Efficiency DH5?-T1 Phage Resistant Cells, Cat. No. 12034-013

MAX Efficiency DH5? Chemically Competent Cells, Cat. No. 18258-012

Library Efficiency DH5? Chemically Competent Cells, Cat. No. 18263-012

MAX Efficiency DH5? F'IQ Cells, Cat. No. 18288-019

MAX Efficiency Stbl2Chemically Competent Cells, Cat. No. 10268-019

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.