MOs were synthesized from Gene Tools LLC, and all of them were stored at −80°C in a concentration of 20 μg/μl (see table S1). Before microinjection, MOs were heated at 65°C for at least 10 min (95°C for 1 min for 3′tR-MOs), snapped on ice, and then diluted to the required concentration. Short RNA oligos/mimetics were synthesized from GenScript Biotech, and all of them were stored at −80°C in a concentration of 0.6 mM (table S2). Digoxigenin-labeled LNA-DNA chimeric oligos were synthesized from GenScript Biotech and stored at −20°C. DNA oligos, including primers, were synthesized by GENEWIZ (table S3). Biotinylated RNA oligos were synthesized using a 5’EndTag kit (Vector Laboratories, MB-9001), which was performed according to the manual. Biotin (Long Arm) maleimide (Vector Laboratories, SP-1501-12) was used for labeling a long-armed biotin group to the 5′ end of RNA oligos (GFP-r1 or 5′tRFlGlu/CTC-mimetic), which was then purified by phenol:chloroform extraction. mRNAs for GFP and NLS-hRNASEH1-GFP were in vitro synthesized using T7 polymerase. The coding sequence of hRNASEH1 was amplified from HeLa cell RNAs using a pair of specific primers. MO or RNA was injected into the yolk of zebrafish embryos at the 1c stage. The injection doses are given in the figures or figure legends.
5 endtag kit
Manufactured by Vector Laboratories
The 5' EndTag kit from Vector Laboratories is a tool used for the addition of a specific DNA sequence to the 5' end of DNA fragments. The kit provides the necessary reagents and protocols to facilitate this process, allowing researchers to incorporate desired sequences at the 5' termini of their DNA samples.
Automatically generated - may contain errors
4 protocols using 5 endtag kit
1
Zebrafish Embryo Microinjection of MOs and RNA
2
Fluorescent RNA Oligonucleotide Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA oligonucleotides (IDT) were reconstituted in distilled water. A 5′ Cy5 fluorescent label was incorporated using the 5′ EndTag kit (Vector Labs) according to the manufacturer's instructions, prior to phenol:chloroform extraction, ethanol precipitation and aqueous resuspension. Concentration of RNA was determined by measuring absorbance at 260 nm. Binding reactions of 10 μl contained 1.0 μl 500 nM Cy5-RNA, 1.0 μl TMEV 2A at concentrations of 280, 140, 70, 35, 17.5, 8.7, 4.4, and 2.2 μM in 10 mM HEPES pH 7.9, 1.0 M NaCl, 5.0 μl 2× buffer (20 mM Tris (HCl) pH 7.4, 80 mM NaCl, 4.0 mM magnesium acetate, 2.0 mM DTT, 10% v/v glycerol, 0.02% w/v bromophenol blue, 200 μg/ml porcine liver tRNA and 800 U /ml SUPERase-In [Invitrogen]) and 3.0 μl distilled water. Final concentrations in the binding reactions were therefore 50 nM RNA, 1× buffer, ∼140 mM NaCl and TMEV 2A at 28.0, 14.0, 7.0, 3.5, 1.75, 0.87, 0.44 and 0.22 μM. All binding reactions were prepared on ice, and samples were incubated at 37°C for 20 min before analysis by non-denaturing 10% acrylamide/TBE PAGE (25 min, 200 V constant). Gels were imaged with a Typhoon FLA-7000 (GE) using the 635 nm laser/R670 filter.
3
RNA-Protein Binding Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Synthetic RNA oligonucleotides (Supplementary Table 5 , IDT) were dissolved in distilled water. RNAs were labelled at the 5′ end with A647-maleimide or Cy5-maleimide conjugates (GE Healthcare) using the 5′ EndTag kit (Vector Labs) as directed by the manufacturer. For each binding experiment, a series of reactions were prepared on ice, each containing 1.0 μL 500 nM RNA, 1.0 μL serially diluted protein at concentrations of 320, 160, 80, 40, 20, 10, 5.0, and 2.5 μM in 10 mM HEPES pH 7.9, 1.0 M NaCl, 5.0 μL 2 × buffer (20 mM Tris-HCl pH 7.4, 80 mM NaCl, 4.0 mM magnesium acetate 2.0 mM DTT, 10% v/v glycerol, 0.02% w/v bromophenol blue, 200 μg/mL porcine liver tRNA, 800 U /mL SUPERase-In [Invitrogen]) and 3.0 μL distilled water. This gave final binding reactions of 10 μL with 50 nM RNA, 1 × buffer, a salt concentration of ~140 mM and proteins at concentrations of 32, 16, 8.0, 4.0, 2.0, 1.0, 0.5 and 0.25 μM. Samples were incubated at 37 °C for 20 min prior to analysis by native 10% acrylamide/TBE PAGE (25 min, 200 V constant). Gels were scanned with a Typhoon FLA-7000 (GE) using the 635 nm laser / R670 filter. Raw, uncropped image data is available in the Source Data file.
4
IRES RNA-Protein Interaction Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wild-type or mutant IRES RNAs were 5’ end labeled with biotin using the 5’-EndTag kit (Vector Labs, Burlingame, CA). Huh 7.5 cells were pelleted from 15 cm plates either untreated or pretreated for 3 hr with 2 mM DTT, lysed by syringe and needle, then clarified and stored at 4°C. Ten micrograms of biotinylated IRES was bound to 50 µL streptavidin-agarose bead slurry, washed, then incubated with cell lysate for 30 min at 37°C. After incubation, beads were washed and used for SDS-PAGE and western blotting.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!