Cloning a zebrafish gene? - (Jan/06/2021 )

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?

The cDNA is equivalent in sequence to the genomic DNA sequence. Because you started with mRNA you only have one DNA strand to work with...

To tag your sequence in particular there are a few different strategies - the most common of which is to put it into a vector which contains a tag already in-frame with the tag. You can, in the absence of a tag in the vector, add the tag to the 5' end of either of the primers so that it is added during subsequent amplification of your sequence.

Depending on how you plan to clone the PCR product (restriction enzyme? (most common), T/A cloning?) you can (also) add restriction sites to the primers. For this you need to find compatible sites in the vector.

Mutagenesis is most likely best done by Qick-Change.

Transformation depends on the bacterial strain you are using. Most people use chemically competent cells for heat-shock transformation(a la Hanahan 1983), but electroporation also works well.

Following this you will need to extract by plasmid DNA isolation.

Zebrafish embryo will be done by micro-injection

As you appear to know little about these processes, I strongly suggest you find a copy of Sambrook et al. Molecular cloning: a laboratory manual. It will be in your local university library, and possibly in your lab somewhere, and someone to learn off, particularly for the zebrafish bit.