So, I am trying to do gene-knockout in Burkholderia ubonensis by using Splicing by overhang extension (SOE) PCR, and I am having problems with designing working primers. This is because our lab doesnt have the sequence of the strain of B. ubonensis we are using. When we design our primers, we relies on the sequence of another strain of B. ubonensis that can be found on Genbank. When we used Clustal Omega to check the alignment of the regions we want to amplified, we see that it's not highly conserved between different strains of B. ubonensis. Because of this, the primers we designed might only work on one strain, but not the others. When I do the alignment, I cant seem to find a conserved region that is big enough (~20bp) for me to design my primers. Does anyone here have any suggestion? How do I design primers that would work between different strains of B. ubonensis?
I don't know what I wrote make any sense. I am an undergrad student, I am new to research. Please forgive me if it's confusing.
As you guys was reading this, I am sure some of you was like "Why doesnt he just sequence the strain he was using?" The answer is we dont have money. I am currently in a very small university that are lacked of resources.
Hi,
In my opinion, you should set sequences by sequencing. After this step you will be able to design highly specific primers.
Good luck!
Unfortunately- sequencing is probably the only feasible way to tackle this problem.
Realising you are tight on budget, only take into account that when considering time and reagents, sequencing might also be the economically efficient method.
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