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Storage of competent cells at temperatures warmer than –70 degrees C, even for a brief period, will significantly decrease transformation efficiency. Cells may still be viable, but will not transform well. A test reaction with a control plasmid, such as pUC19, can be performed to determine transformation efficiency of cells stored in this way. This will help you determine whether or not they are still usable for your experiments.

We do not recommend storage of competent cells in liquid nitrogen, as this will harm the cells. Additionally, the tubes that the competent cells were supplied in may not be able to withstand this temperature, leading to cracking or breaking.

XbaI cutting site is a Dam-methylation sensitive restriction site. TOP10 is a dam(+) strain, which means it expresses 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, Taq I, Xba I, BspH I, Nde II, Nru I
  • Dcm: Ava II, EcoO 109 I, EcoR II, Sau96 I, ScrF, Stu I, Aat I, Apa I, Bal I, Kpn I, ISfi I

If you are using a mcr/mrr(+) competent cell strain, cellular enzymes may be recognizing eukaryotic methylation patterns on the yeast genomic DNA and deleting or rearranging it. Try a mcr/mr(–) strain such as Top10, DH10B™, or OmniMAX™ 2.

There are a few conditions that can lead to this: SOC medium or other media used when plating was contaminated, DNA was contaminated with amp-resistant microbes, old plates with degraded amp were used, or the competent cells themselves were contaminated.
The F’ episome in TOP10F’ has a lacIq marker, which over-expresses the lac repressor. IPTG must be added to LB plates along with X-gal to see beta-galactosidase expression and blue color in this strain. TOP10, on the other hand, does not require IPTG for blue/white screening.

We recommend trying the following:

  • Carry out the pUC19 transformation control; this gives you information about the performance of the cells.
  • Check plates for expiration and correct media used (LB/agar).
  • Confirm that the correct antibiotic and concentration was used.

With any strain, the first thing to try would be to lower the growth temperature of the culture to 30 degrees C or even lower (room temperature). Slower growth will generally allow E. coli to tolerate difficult sequences better. If reducing the growth temperature doesn’t help, you may want to consider using a competent cell strain such as Stbl2™ or Stbl4™ cells, which have been shown to accommodate this type of sequence better than other strains in the same conditions.

When electroporating cells at high voltage in conductive buffers, arcing may occur. MgCl2 and PO4 in particular are very conductive.

Some suggestions:

  1. Keep ionic strength of cloning reactions to a minimum. If reaction buffers contain high salt, dilute DNA sample in water or TE buffer before electroporation.
  2. Minimize addition of conductive ions. The volume of DNA solution should not exceed 5% of the total reaction. Example: 2 μl DNA per 40 μl of cells
  3. Be sure no air bubbles are present.
  4. Make sure the electrical contacts are clean and tight. Wipe away any condensation on the outside of the cuvette.
  5. For best results, the cells should be aliquoted into the bottom of the gap. Tap the cuvette gently to help the cells settle to the bottom.
While there is no specific strain that works better with large plasmids, it is important to focus on transformation efficiency. For larger plasmids, chemically competent cells with highest efficiency are suggested, such as OmniMAX™ 2, TOP10, etc. We would recommend using an electrocompetent cell strain with plasmids larger than 20 kb for best efficiency.
Check the genotype of the cell strain you are using. Our Gateway® destination vectors typically contain a ccdB cassette, which, if uninterrupted, will inhibit E. coli growth. Therefore, un-cloned vectors should be propagated in a ccdB survival cell strain, such as our ccdB Survival™ 2 T1R competent cells.

Here are a few suggestions:

  • Small fragments/linkers are cloning in to your vector instead of your insert. To correct this, gel-purify the insert before ligation.
  • Ensure that the correct concentrations of X-gal and/or IPTG (if vector contains the lacIq marker) are used.
  • If spreading X-gal and/or IPTG on your plate, allow sufficient time for the reagents to diffuse into the plate.
  • Incubate your plate for a longer period to ensure full color development.
Ensure that you are using the correct antibiotic at the appropriate concentration. Additionally, make sure the antibiotic is not expired. If colonies exhibit unexpected morphologies, contamination could be a factor. Check your S.O.C. medium and LB growth medium.
Subcloning DH5α™ cells are a compatible strain, but you will get lower efficiency (10e6 vs 10e9) and therefore risk getting fewer clones. Top10F’ is also compatible, but if blue/white screening is performed, IPTG along with X-gal will be needed for detection due to the expression of the lacIq repressor present in cells containing an F’ episome.