Rna clean and concentrator kit rcc

The rbbp4 cDNA was cloned into the expression vector pT3TS. A 1 μg linearized vector was purified with the PureYield Plasmid Miniprep System (Promega, A1223) and used as template for in vitro synthesis of capped mRNA with the Ambion mMessage Machine T3 Transcription Kit (Thermo Fisher, AM1348). In vitro synthesized mRNA was purified with the RNA Clean and Concentrator Kit RCC (Zymo, R1013). Single‐cell zebrafish embryos were injected with 100 pg or 300 pg rbbp4 mRNA. At 24 hpf, the embryos were incubated for 5 hours with 100 μm Camptothecin (Sigma‐Aldrich, C9911) or DMSO as a control. Embryos were incubated in 10ug/ml acridine orange (Thermo Fisher Scientific, AC423340010) in embryo media for 30 minutes. acridine orange‐labeled living embryos were rinsed with embryo media and immediately imaged on a Zeiss Discovery.V12 stereomicroscope using a Cannon Rebel digital camera and EOS Utility software.

For synthesis of Cre and Dre mRNAs, the expression vectors pT3TS-Cre (Clark et al., 2011 (link)) and pT3TS-Dre were linearized with SalI (New England Biolabs, R0138S) and BamHI (New England Biolabs, R0136S), respectively. 1 μg linearized vector was purified with the PureYield Plasmid Miniprep System (Promega, A1223) and used as template for in vitro synthesis of capped mRNA with the Ambion mMessage Machine T3 Transcription Kit (Thermo Fisher, AM1348). In vitro synthesized mRNA was purified with the RNA Clean and Concentrator Kit RCC (Zymo, R1013). A total of 12.5 pg Cre or 15 pg Dre mRNA was injected into one-cell stage embryos to promote recombination-mediated inversion of the UFlip cassette at lox or rox sites. UFlip cassette inversion in Cre or Dre injected 3 dpf larvae was confirmed by digestion of individual larvae in 50 mM NaOH at 95 °C for 30 min and neutralization by addition of 1/10^th volume 1 M Tris-HCl pH 8.0. Genomic DNA/UFlip cassette 5’ and 3’ junctions were amplified by PCR with gene specific and UFlip primers listed in Table 4, followed by direct Sanger sequencing.