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?