Troubleshooting forum. Molecular biology techniques Q&A.

Site-directed mutagenesis: colony growth This month's question from the Molecular Biology Forums (online at molecularbiology. forums.biotechniques.com) comes from the " General Methods " section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but do not represent endorsements by, or the opinions of, BioTechniques. Why don't I get many colonies following transformation of the DpnI-digested PCR product? (Thread 21770) Q I have been working on site-directed mutagenesis (SDM) for over a month, but still haven't been able to get the mutation I need. I used Fermentas Pfu polymerase to amplify my vector with insert and saw two bands following the PCR. I then set up a DpnI digestion of the PCR products using 3 µL PCR product, 1 µL DpnI, 1 µL buffer, and 5 µL water to make a total volume of 10 µL. I digested the product for 90 min at 37°C. When I ran a gel following digestion, I didn't see any bands at all, but I transformed the JM109 cells anyway using 1.5 µL of the digested product. After overnight incubation at 37°C, I had only a single colony. The colony did not grow in Luria Bertani (LB) broth with ampicillin or on an LB plate. I think this colony must contain my mutant, but what went wrong? Why won't it grow? A When I do SDM, I add the DpnI and its buffer directly into the PCR reaction. I don't know if that will make much difference for your results, but I thought I would let you know since that is easier. I also don't worry much about checking each step on gels since I am most concerned about the end results. You mentioned that you got only a single colony after incubating at 37°C overnight. Were those cells incubated with LB only or with LB plus antibiotic? If you added the antibiotic, did you incubate at 37°C for 1 h without antibiotic first? I suggest you check your competent cells. Did you include any controls? A I recommend that you use the Stratagene kit; it makes the procedure quite simple. Adding 1 µL DpnI to 3 µL PCR product seems like a lot to me. I usually add 1 µL DpnI straight into my PCR reaction as described in the previous post, but I don't add the DpnI buffer. I incubate this …