I have been working with the Heat Shock Protein 70 (Hsp70) developing a CRISPR/Cas construct to replace Hsp70 with Hsp70-GFP. After microinjecting optically clear zebrafish embryos, I visualized them at 72 hpf and saw only a handful of "positives" under a fluorescent microscope. However, when I digested the embryo and ran a PCR to amplify the GFP, I had many more bands in my "negative" samples than I did in my "positives." Any suggestions for why I might be seeing either a false GFP in my PCR, or not seeing the GFP present under the microscope? Thanks.
If you see some positives, then the sequence should be correct, you still might want to make sure you have sequenced your full insert (Hsp70-GFP).
Are the "many" bands from your negative samples of the right size?
Can you confirm proper expression in a cell-line?
Can you do a western blot with anti-Hsp70 and anti-GFP antibodies?
Well....the fact that the DNA is there does not mean it's expressed! if you've injected it it should be in virtually all your embryos...but from the construct to the expression...that's a different story. Under the microscope you see the fluorescent protein....with PCR you identify the construct....there's a translation step in the middle. Also what do you see in your NO template control after PCR?
It is possible that the GFP is being expressed in many of the embryos, but that it's being expressed at such a low level that you're not able to visualize it.
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