Hello!
I am microinjecting Tol2 and UAS:mScarlet into zebrafish, and I was looking up what the optimal concentration of Tol2 and plasmid I should be injecting. There were a couple protocols that have 0.4M KCl included in their injection mix, and I was wondering if anyone knew why?
When I would inject, I would only use ddH2O, phenol red, Tol2, and my plasmid.
Also, if anyone has used Tol2 before, what concentration (of both Tol2 and plasmid) did you inject?
Thank you!
KCl is primarily to keep the osmolarity of the embryo and to 'mask' charges, when injecting nucleic acids
KCl is primarily to keep the osmolarity of the embryo and to 'mask' charges, when injecting nucleic acids
Concentrations:
Expression constructs were tested by injection of plasmid DNA, with or without transposase RNA, into the cell at the one-cell stage. For verification of the IRES constructs (Fig. 2), approximately 75 pg of DNA was injected per embryo. For tests of the Tol2 transposase (Figs. 3, 4), either 25 or 30 pg of DNA was injected (see Figure Legends for details) ± 25 pg of transposase RNA
http://onlinelibrary.wiley.com/doi/10.1002/dvdy.21343/full
You can see
https://www.researchgate.net/deref/http%3A%2F%2Fonlinelibrary.wiley.com%2Fdoi%2F10.1002%2Fdvdy.21343%2Ffull
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