Troubleshooting Transformation

Reproduced from Life Technologies, Inc.

Shanta Dube
Technical Services
Life Technologies, Inc.
Rockville, Maryland 20849

If you are getting low transformation efficiency with chemically competent cells:

• There were impurities in the DNA. Remove phenol, protein, detergents, and ethanol, by ethanol precipitation or GlassMAX DNA Isolation Systems.
• There was excess DNA. Use no more that 1-10 µg of DNA in <5 pl volume/100 pl of cells.
• The cells were handled improperly. Thaw cells on ice, and use immediately. Refreezing can decrease the efficiency. Do not vortex cells.
• Nonoptimized tubes were used. Use 17 x 100-mm polypropylene tubes. The heat shock step is calibrated to the size and material of these tubes. If different tubes are used, the heat transfer to the cells may not be optimal.

If you are getting low transformation efficiency with electrocompetent cells:

• Salts and buffers inhibited electroporation and may have caused arcing. Use DNA in water or TE (10 mM Tris- HCl, 1 mM EDTA).
• The apparatus settings were not optimal. When using the GIBCO BRL CELL-PORATOR~ System, use 0.15-cm gap chambers and 400 V on the CELLPORATOR unit, 330 pF; low ohm setting; fast charge rate; 4,000 ohm resistance on the Voltage Booster. On the GIBCO BRL E. coli CELL-PORATOR Apparatus, use 0.15-cm chambers with the medium setting.
• The microelectroporation chambers, cells, and DNA were not chilled on ice. Also, equilibrate the control compartment of the chamber safe or single safe to 4_C prior to electroporation.
• An excessive volume of cells was used. Use <25 pl and place the cells between the indented bosses.

If you are getting all white colonies, but your clone does not have an insert:

• IPTG was not used with vectors containing the lacIq marker. Spread 30 µl of 100 mM IPTG solution on the surface of the plate.
• The X-gal did not diffuse into the agar properly. Spread 50 pl of 2% X-gal on the surface of the plate or add X-gal to cooled medium before plating at a final concentration of 50 µg/ml. Plates with X-gal should be stored at 4_C in the dark. Do not use plates with X-gal in agar that are >4 months old.
• The color did not develop fully. Incubate at 37_C for 12 to 16 h.
• Small linkers and adapters ligated into the vector. Gel purify the insert and vector prior to ligation.
• The vector used did not allow for a-complementation. Check that the vector contains the a-peptide of the lacZ (ß- galactosidase) gene.

If you are getting all blue colonies with recombinant DNA:

• The insert was cloned in frame with the a-peptide. Another screening method, such as PCR, is necessary to find recombinants.
• The E. Coli strain used had an intact ß-gal gene. Use an E. Coli host containing the lacZ6M15 partial deletion of lacZ that allows for a-complementation.

If performing large-scale plasmid preps, you have cell death or low cell growth with decreased plasmid yield:

• The insert produced a protein that was toxic to the cells. Decrease transcription of the protein, by adding 20 to 30 mM glucose to terrific broth. Decrease plasmid copy number by changing to a non-pUC-based vector or by incubation at 30_C instead of 37_C.

If you see an opaque colony:

• The protein/RNA transcripts being expressed from the doned insert altered the morphology of E. cold colonies (opaque, instead of translucent). This is gene dependent [see Barik, S. (1997) BioTechniqsfes 22.1, 112].

If your restriction endonuclease is not digesting DNA:

• The restriction endonuclease may be dam and dcm sensitive. If so, propagate the DNA in a dam and dem minus strain, such as DM1 Cells (Cat. No. 18268).

If you get extra bands that migrate faster than supercoiled DNA on an agarose gel and are resistant to cleavage by restriction endonucleases:

• The DNA was irreversibly denatured. This has been observed when plasmids were isolated by the alkaline Iysis method. To minimize denaturation, use 0.1 N NaOH instead of 0.2 N NaOH and decrease the time of NaOH exposure.