Células competentes ElectroMAX™ Stbl4™

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.

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.

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.

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.

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.