Cloning a zebrafish gene

[KubaK]

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?

[CharonY]

4 hours ago, KubaK said:

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?

I am not sure I understand the question is- in a standard PCR you obviously amplify both strands of your cDNA. So generally you would just create suitable forward and reverse primers with the tags you need. Or is the question more about primer design in general.

I will add that typically you should talk to your supervisor about the project. Often there are standard practices in place that one should adhere to.