Troubleshooting Guide

1. Following restriction enzyme digestion and electrophoresis, the number of bands is as expected but the sizes of one or more bands are not. This is most likely due to inaccurate information of the restriction enzyme site in the vector or insert DNA. Try to be careful when analyzing the restriction enzyme map, cutting site and sizes of DNA fragments.

2. Many unexpected bands show up after a single restriction enzyme digestion of the vector or insert DNA. It seems that the conditions set up for restriction enzyme digestion are not appropriate. As a result, the enzyme activity is not specific, but has cut DNA into pieces rather randomly. Make sure that the reagents and temperature are optimal for the enzyme.

3. Compared with undigested DNA, incomplete restriction enzyme digestion occurs. The solution is to increase slightly units of the restriction enzymes or increase digestion time for one more hour. Additionally, try to use fresh enzymes.

4. No colonies are observed following antibiotic selection. This is a complicated situation. Most likely, no ligation occurred between the vector and insert DNA. All the DNA molecules transformed into bacteria are linear DNA degraded rapidly in bacteria. Or, extremely high concentration of antibiotics such as ampicillin may be used in LB plates, which will kill all the bacteria, including transformants. Try to increase the efficiency of ligation and make sure that an appropriate antibiotic is utilized in the LB plates.

5. A large percentage of colonies is due to self-ligation. This is a common problem when ligation occurs between the single-restriction, enzyme-digested vector and the insert DNA. The effective solution is to increase the units of alkaline phosphatase to remove the 5' phosphate group from the vector prior to its ligation with insert DNA. Whenever possible, try to choose two unique enzymes that can generate two incompatible sticky ends to eliminate self-ligation.

6. The size or pattern of digested DNA does not match those of the vector and insert used for ligation. This may be due to mutation occurring during transformation or replication of plasmid DNA in bacteria. If this happens, there is no effective way to solve the problem except for eliminating the bacterial colonies.

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