Full-length clone of mouse Mklp1 (MGC: 54492) was purchased from Open Biosystems and amplified for insertion into pIVT-eGFP or pIVT-mCherry 60 (link). Gap43-eGFP was a gift from Dr. Greg FitzHarris (U. of Montreal). To generate cRNA of Eb3-egfp61 (link), Mklp1-eGfp, Mklp1-mCherry, and Gap43-eGfp, plasmids were linearized and transcribed in vitro using mMessage mMachine T7 or SP6 kit (Ambion, Cat# AM1344) according to manufacturer’s protocol.
cRNA was purified using SeraMag Speedbead (Sigma Aldrich, Cat# GE65152105050250) nucleotide purification method previously described 62 (link). Briefly, in vitro transcription reaction solution was brought up to 150 μl and mixed with 100 μl of magnetic beads and let stand for 5 minutes. Beads were then pelleted using a magnetic stand and washed with 80% ethanol. cRNA was eluted using 20 μl nuclease-free water and stored at −80° C.
cRNA was purified using SeraMag Speedbead (Sigma Aldrich, Cat# GE65152105050250) nucleotide purification method previously described 62 (link). Briefly, in vitro transcription reaction solution was brought up to 150 μl and mixed with 100 μl of magnetic beads and let stand for 5 minutes. Beads were then pelleted using a magnetic stand and washed with 80% ethanol. cRNA was eluted using 20 μl nuclease-free water and stored at −80° C.
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