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  • FAQ: What are some other problems that should be considered when trouble shooting a transformation problem?

    • The cells are not viable. Run the controls listed below and obtain new cells if needed.
    • The cells are not competent. Run the controls listed below and obtain new cells if needed.
    • The recombinant protein is not well tolerated by E. coli. Try making a fusion with maltose binding protein using the pMAL System (NEB# E8000S). Try another expression system that doesn't involve E. coli.
    • The ligated DNA included inverted and tandem repeats selected against by E. coli. Remove the repeat sequence if possible. Try another expression system that doesn't involve E. coli.
    • The insert DNA was taken from mammal or plant and contains methylated cytosine which is degraded by many E. coli strains. Use a strain deficient in mcrA, mcrBC and mrr.
    • Construct is too large (>10,000 bp) for transformation into chemically competent cells. Use electroporation.