how to mutant the gene on the genomic DNA? - (Nov/23/2005 )
I want to mutant the gene on the genomic DNA using one crossover. So I constructed the plasmids which have part of the gene I want to mutant, and part of the next gene, and ligated with the spectinomycin gene for selection. Then transform into the bacteria. And I got a lot of colonies, but after isolating genomic DNA and PCR, I have not got any positive colonies. Does any one have any good idea? Or all of you will pick up hundreds of colonies to do PCR before getting the positive one. It made me mad. Thanks for your help.
when you say you have not got positive colonies, do you mean you are getting only single cross-overs? I had to screen about a hundred before I for a double cross-over.
Yes -- there's something missing here. Are you using a suicide vector? On what basis are you selecting your transformants?
You can't mutate a gene on the chromosome with a single crossover, you need a double crossover to remove the wildtype copy.
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are you looking for gene disruption or specific mutations? if you are aiming at gene knock out, try preparing a disruption vector having a marker inserted between gene to be disrupted cloned in a plasmid then perform transformation. select transformants based on the marker, then perform southern blot to check if it has inserted at the right place via honologous recombination.
do mail back if this is what you were looking for.
best of luck.
I wanted to do gene disruption and I did the double cross-over mutation before, using SacB selection, but it did not succeed after picking up a lot of colonies. That is why my boss let me do just single cross-over mutation, although that will disrupt the gene down. But it does not matter for our case. My problem is that I did not get any one cross-over mutation after picking up almost 50 colonies. That made me crazy. Thanks, guys.
It looks like you are not getting any recombination. Firstly, what strain are you using? Secondly, how large in your homologous region? Thirdly, have you checked where your constructs are integrating? Finally, do you know if your knock out is lethal- if it is you won't be getting too many recombinants?
I used Actinobacillus actinomycetemcomitans, one oral bacteria. And my homologous region was about 1300bp. I am sure my constructs are integrating, and the knock out is not lethal to the bacteria.
If the plasmid cannot replicate, and it carries the selective marker, then there is no way for you to get any colonies without a cross-over event. That being the case, if the colonies you're screening do not carry the plasmid in the expected place, it's crossing into the chromosome elsewhere. The question now is one of experimental design -- how is the plasmid crossing into the cromosome at some place other than the targeted gene, and how can I stop it from doing that?
How are you screening these colonies? What portion of the gene have you cloned for the insetional mutation strategy? Are you sure your colonies are not meridiploid and throwing off PCR screening? Have you considered checking them by Southern blot?
I think you need to tell us a lot more about what you are trying to do before we can really help. What gene, how you are transforming, how you art screening, etc.
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