I want to insert the km resistance gene into the ampc gene in E.coli. I want to do this with Datsenko and Wanner method. I amplify the km gene from the pkd4 plasmid with a 40 base tail at the ends. Then transform this insert into E.coli cells containing plasmids pkd20 and pkd46 by the CaCl2 method. After waiting for 45 minutes on ice for transformation, I expose it to 42 degrees in a water bath for 2 minutes and keep it on ice again for 2 minutes. I then incubate at 30 degrees for 1 hour in SOC medium (without arabinose) and then spread on LB agar containing KM.
Usually, many colonies are formed within 2 days. When I PCR these colonies from the outer part of the gene, a PCR product of similar size to the wild type is formed.We have the same problem for many genes in the lab. All of the colonies we get are false positive.
I also get false positives when I use the chloramphenicol resistance gene instead of kanamycin.
Colony PCR will give you false positives if you are using the original plate from your transformations. There will be a lot of plasmid molecules that were not transformed into any bacteria just sitting on the agar and PCR is sensitive enough to amplify from that.
Streak any possible colonies onto a new plate and set up PCR from the new plate.
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