My goal is to be able to integrate a plasmid containing the GFP construct where there will be the desired cleavage from the Cas9.
To do so, i did as described in the paper of Auer T.O. et al (Genome research 2014): I have been trying to engineer a plasmid using a Topo-TA vector that will be inserted with my guide RNA and a GFP construct. I manage to have the blunt GFP contruct that I tried to ligate to my guide RNA. After I tried to transform the TOPO-TA vector. However for now i haven't been successful at all as i didn't manage to have any efficient transformation.
Does anyone as already tried to do this type of construct and will have some input?
Hi. As i find you want to integrate your gene with GFP and then colon it into the topo/TA vector. why don't you use gibsson assembelly method, for integrate your fragments, that does not required any restriction enzyme? With this method you are able to create too many mutations and any thing that you want.
i hope that is helpful
Does addgene.org already have what you need? Check our crispr resources or call for help in finding a plasmid.
addgene.org/crispr
I actually found what i needed on sigma but it seems that i manage by PCR insertion in a small plasmid to duplicate what they sell.
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