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KubaK

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    Cloning a zebrafish gene

    KubaK posted a topic in Biochemistry and Molecular Biology
    Hello! I need a bit of help with my Msc research. I've identified a mutation that I want to check in zebrafish. Workflow is: 1. RNA isolation and reverse transcription (oligo(dT) primers) 2. Here i have a problem. I want to tag those genes with HA-tag, but i dont know how to design primers valid for cDNA amplification. How to amplify this correct strand of cDNA i want to check? 3. Put it in a pCS2+ plasmid. 4. Mutagenesis of a specific nucleotide. 5. Put it into the bacteria, isolation and shooting into the zebrafish embryo. How do I design those primers specific for cDNA? Is there any spoecific way, or just normally as in regular genomic PCR? Also during reverse transcription should I use oligo(dT) and random hexamers mixture?

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