In need to clone my oligo into my plasmid digested at the BbsI site. However, my plasmid doesn't have the BbsI site. Do I need to add this site to the plasmid? If so, how do I go about doing this?
I recommend you use Klenow or T4 DNA pol to generate blunt ends, then you can use an enzyme that generated blunt ends in the plasmid,
Hi Matthew,
SIlvia Mora's answer should be my choice if I am in your current situation. Don't forget to do dephosporylation in the blunted end plasmid to avoid self-ligation. Or I will add unique restriction site (that the plasmid has) in the end of the insertion sequence (oligo) by PCR, instead of adding BsbI into the plasmid.
If you want to know how to add BsbI into the plasmid,
1. Try this
https://www.addgene.org/plasmid-protocols/annealed-oligo-cloning/
Or,
2. If your plasmid is no longer than 4kb* I recommend you to linearize your plasmid and do PCR using hi fidelity polymerase amplifying the whole plasmid. Both primers used should be tagged by BsbI sequence plus additional 5-10 nucleotide. purify your pcr product, digest by BsbI, self-ligation and do cloning to amplify this plasmid. Contact me if you need detail protocol for this.
*) In my experience, DNA longer than 4kb will risky in nucleotide substitution when amplified by PCR even using hi fidelity polymerase
Cheers,
Kurnia
This is an outdated way of doing it. It will be more efficient to learn a restriction-independent method like Gibson's Assembly, especially if you have to do this kind of thing often.
BbsI have a cutting site GAAGAC(2/6)^. so there will be a 4 nucleotide overhang of random sequence. why on earth you want to use this enzyme to clone your oligo into plasmid that not at all having this cutting site? please clear the situation you are in, to get a helpful reply.
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