Dear all,
I need to genotype some zebrafish larvae at 3-4dpf. I currently tried to perform it as I do it for the adults, but it did not work. Basically:
1. Fin clip and add the fin to a 96 well plate
2. Extract gDNA by adding 40uL of lysis buffer + prot K
3. Incubate for 1h at 60C and 10mins at 95
4. Perform KASP assay as stated in LGC Group.
The issue is that I think I can't get enough DNA with the 3dpf, as the fin clip is much smaller than when I do it with the adults.
Does anyone have any idea how to approach this?
Does anyone have access to the paper by Kosuta C. et. al., 2018 with the title " High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping."? I think that article might help but I don't have access.
I'd really appreciate your help.
Thanks,
Raquel
Article High-throughput DNA Extraction and Genotyping of 3dpf Zebraf...
if you are not working with much dna then you might consider using nested or semi nested pcr to increase the amplification of the small amount that you do have
I had good luck getting DNA out of individual nematode worms using a similar DNA extraction protocol WITH the addition of 5-10 freeze-thaw cycles after adding the proteinase K and lysis buffer. Basically, put your samples in the -80 freezer until solid, thaw at room temp, and repeat 5-10 times. It helps break open the cuticle and cells. Might be worth a try with your fin clippings.
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