Also, does anyone have an idea how big your insert can be in a 6 kb vector when you use the gibson assembly as the cloning method to create a midigen and where you only design a forward primer at the beginning and a reverse primer at the end of your insert?
I have now developed a midigen with an insert of 4 kb (NF1 gene exon 20 to 24 with introns) in a pSPL3 vector of 6 kb, so together that is 10 kb. I did this using the Gibson Assembly cloning method. I designed a forward primer in intron 19 and the reverse primer in intron 24. For sequencing, I designed new primers to divide the 4 kb piece into pieces.
I am trying to develop midigene to study larger splicing effects with the mini/midigen exon-trap assay.
Minigenes can also refer to a truncated but still functional gene that usually has the minimal promoter, absent or reduced introns and a fragment of the CDS like the Drosophila mini-white gene.
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