I have amplified 500 bp up- and downstream of my target gene and placed a KanR for selection between both 500bp fragments. Is that correct?
Now, I want to use pGMT-t vector (Promega) as a shuttle, as most papers i read do it.
How do i insert my construct into the vector resp. must the vecter be linearized before transformation in (naturally competent) S. pneumoniae? If so, how do I apply it ?
Dear Stefan
Please check this: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3958881/
Best Wishes
Amin
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3958881/
Stefan, I have used this protocol to make knockouts in pneumococcus several times. https://www.ncbi.nlm.nih.gov/pubmed/10188240
It is not necessary to put your construct into a vector. The homologous recombination event after transformation should work just fine with a purified PCR product. I would suggest doing a test transformation first with serial dilutions of the the DNA you are adding since transformation efficiency depends greatly on the strain of pneumo you are using. (If it's an encapsulated strain e.g. more DNA will need to be added).
Also, you will want to incubate with the competence-stimulating peptide for 15 minutes at 37˚ then add your DNA and incubate for an hour. Good luck
-M
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