I am trying to replicate the methods in this paper:Article Purification of an Intact Human Protein Overexpressed from I...
) to design/order an HDR donor with the following tags: GFP-Hygr, CMV-promoter, and SP-FLAG.I have heard using plasmids is not the best option for CRISPR based assays and not sure if I can design a custom vector with all those tags withiout using a plasmid. I am new to CRISPR and appreciate any suggestions/thoughts.
The goal of the assay is to insert a strong propmoter (CMV) via CRISPR into an endogenous gene (RELN) for reeling overexpression in HEK293 cells.
https://blog.addgene.org/crispr-101-homology-directed-repair is a good start.
You haven't said how long your proposed HDR template is and how much you would need of it - but really, why mess around cloning anything below 1500 bp together yourself, just order the doublestrand sequence as gBlock, m-block, DNA Fragments (a couple different companies that do the same, not exhaustive list!). It's way faster and cheaper to do it this way, rather than spending weeks cloning three different bits together.
Then either use the gBlocks directly as HDR or order the sequences with compatible ends to stick into a plasmid and then use the plasmid as template. If you think about which restriction enzymes to add&use, you can get the insert cut out without extra sequences.
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