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| 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:
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).
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:
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