Hi everyone!
I would like to knockout a gene in the genome of Bacillus. To do that I tried to make a construct that has 1) a backbone, 2) a kanamycin resistance cassette, flanked by 3) the upstream and 4) downstream regions of the target gene. The aim is to replace the gene with the cassette by the process of homologous recombination.
So, I amplified these pieces (1-4) and went on to "connect" them with the Gibson reaction (the primers have homology with the piece they should connect with). But the Gibson product, when I tried to digest it with a restriction enzyme to check if the ligation has taken place didn't give me any product at all (although the con/on in Nanoprod of the Gibson product was ~1000ng/uL). I did the calculations for the Gibson as follows:
And then, I divided by the concentration of my products to find the uL I should add to have a mixture of 10uL total.
Total: 6 + 5.85 + 0.51 + 0.74 = 13.1 uL of mix.
And I added from this 10uL to 10uL of Gibson reaction mix. It didn't work. I tried to modify the neoR quantity changing 3 to 2,5, but still, it didn't give any results.
Any ideas on what might have been wrong? Should I modify something of the above? Should I change my method altogether? (Is there any better method for knocking out a gene?)
Thank you very much!
Hello, To knock out the gene is important to use an appropiate selection marker and antibiotic resistance genes are frequently used for this aim. The Chloramphenicol Acetyl-Transferase = Chloramfenicol Resistance because other genes such as that conferring Kanamycin resistance frequently produce a lot of false positive mutants because natural resistance of strains, then you turn to select with very high concentrations of Kanamycin.
The bes way is CRISPR. Mainly CRISPR Red λ-Red method.
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