Having difficulties with your experiment?

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General

Problem Possible cause Recommendation
Few or no colonies Poor transformation efficiency Check competency of your Life Technologies™ E. coli with the pUC19 control plasmid provided with them. See also Troubleshooting your transformations.
Ligation reaction did not work properly Make sure vector or insert has a 5’ phosphate
Check ligation reaction components by ligating lambda DNA/Hind III markers and comparing to unligated markers on a gel. Marker bands should disappear and a high molecular weight smear should appear. You can also try ligating single cut vector.
Check for inhibitors in insert and vector DNA by adding some to a lambda DNA /Hind III marker ligation. If markers ligate alone but not when other DNA is present, the other DNA contains an inhibitor. Purify your DNA prior to ligation
Be sure that the RE used to prepare the vector and insert has been removed by phenol extraction, buffer exchange, or at least inactivated.
Incomplete digestion of vector and/or insert Run digestions on agarose gel to verify cleavage. If digestion is incomplete, re-digest or gel purify digested fragment.
If adding restriction sites via PCR primers, make sure there are enough nucleotides upstream of the restriction site to allow proper cleavage(it is RE dependent, at least 6 is recommended)
Make sure you are using proper digestion buffer for the RE.
Incompatible ends Make sure the ends generated are compatible (blunt and blunt or compatible overhangs)
Too high concentration of antibiotic was used Make sure you use the correct amount of antibiotic.
Wrong antibiotic was used Make sure to use the antibiotic whose resistance marker is carried on the vector.
Damaged or nicked DNA Minimize DNA exposure to UV light during gel purification
Many colonies not containing the insert Vector self-ligation Vector dephosphorylation was not efficient. Confirm by ligating and transforming dephosphoylated vector. Repeat CIAP treatment of vector.
Undigested vector still present Confirm by transforming digested unligated vector. Gel purify the digested vector to be used in the ligation.
Insert is toxic to the cell Try growing the cells at a lower temperature (30 degrees C or room temperature). Colonies will take longer to appear.
Try a different strain (example Stbl 2 E. coli)
Many colonies not containing plasmid Too low concentration of antibiotic was used Make sure you use the correct amount of antibiotic.
Antibiotic has degraded Do a mock transformation (no vector) to be sure the antibiotic selection is working

If the insert is potentially toxic to the host cells, here are some suggestions that you can try:

  • After transforming TOP10 or DH5α cells, incubate at 25-30 degrees C instead of 37 degrees C. This will slow down the growth and will increase the chances of cloning a potentially toxic insert.
  • Try using TOP10F’ cells for the transformation, but do not add IPTG to the plates. These cells carry the lacIq repressor that represses expression from the lac promoter and so allows cloning of toxic genes. Keep in mind that in the absence of IPTG, blue-white screening cannot be performed.
  • Try using Stbl2 cells for the transformation.

Digestion

You can perform a sequential digestion where you digest with the first enzyme, clean up the reaction (e.g., with agarose gel/spin column), then digest with the second enzyme. Alternatively, if you are going from a low salt buffer to a high salt buffer, you can perform your first digestion, then add the salt and second enzyme for the second digestion and skip the clean-up step.

Some restriction enzymes demonstrate “star activity”, where they cleave sequences similar to the defined sequence they recognize. The following conditions may contribute to star activity (different restriction enzymes will be affected differently by each condition).

  • Incorrect buffer conditions: Make sure you use the recommended buffer for single or double digestions.
  • Incubation time is too long: Use the minimum time needed for complete digestion.
  • Organic solvents (ethylene glycol >45%, dimethylsulfoxide(DMSO) >8%, ethanol >12%, dimethylacetamide, dimethylformamide (DMF), sulphalane)
  • High glycerol (>5%). Life Technologies™ restriction enzymes (REs) are supplied in 50% w/v glycerol, therefore the RE should not comprise more that 10% of the final volume of the digestion.
  • High enzyme/DNA ratio (>25 U/μg): Use the minimal amount of enzyme necessary, this will also prevent carrying too much glycerol into the reaction.
  • Mn++ or other divalent cations substituted for Mg++: Make sure you use the recommended buffer.
  • High pH >8.5: Make sure you use the recommended buffer.

The Xba I cutting site is a Dam methylation-sensitive restriction site. E. coli strains that are dam(+) strains, like TOP10, express 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 (Nde II), Taqα I, Xba I, BspH I, Nru I
  • Dcm: Ava II, EcoO 109 I, EcoR II, Sau96 I, ScrF, Stu I, Aat I, Apa I, Ban I, Sfi I