Apppg

Manufactured by New England Biolabs

ApppG is a dinucleotide triphosphate that can be used as a substrate for enzymatic reactions involving RNA or DNA synthesis. It is a chemically synthesized compound that can be utilized in various molecular biology applications.

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Lab products found in correlation

M7gpppg cap analogue, by New England Biolabs (1 mentions) Cell disruption bomb, by Parr (1 mentions) Scriptcap m7g capping system, by Illumina (1 mentions) Vaccinia capping enzyme, by New England Biolabs (1 mentions) Rna purification kit, by Macherey-Nagel (1 mentions) T7 rna polymerase, by New England Biolabs (1 mentions) M7gpppg cap analog, by New England Biolabs (1 mentions)

4 protocols using apppg

In Vitro RNA Transcription and Purification

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Transcription templates were generated by PCR amplification from the plasmids pUC19-TIE. The amplified templates were used for in vitro transcription with recombinant T7 RNA polymerase in the presence of either m⁷G_pppG cap analogue or non-functional cap analogue A_pppG (New England Biolabs). To check RNA integrity, an aliquot was mixed with formamide dye and loaded on 4% denaturing polyacrylamide gel. The RNA was visualized under UV light after ethidium bromide staining. To eliminate unincorporated nucleotides, the RNA sample was loaded on a gel filtration Sephadex G25-column (Pharmacia Fine Chemicals), proteins were then eliminated by phenol extraction, and the RNA transcripts were precipitated with 0.25 M NaCl in ethanol. After centrifugation, RNA pellets were dried and resuspended in autoclaved milli-Q water. The concentration of purified RNA samples was determined by absorbance measurement at 260 nm.

Alghoul F., Laure S., Eriani G, & Martin F. (2021). Translation inhibitory elements from Hoxa3 and Hoxa11 mRNAs use uORFs for translation inhibition. eLife, 10, e66369.

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Cell-Free Translation from RACK1-Silenced Extracts

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In vitro translation competent extracts were prepared from control or RACK1-silenced S2 cells as described in (Wakiyama et al., 2005 (link)). Briefly, cells were resuspended in lysis solution [40 mM Hepes–KOH (pH 8), 100 mM potassium acetate, 1 mM magnesium acetate, and 1 mM dithiothreitol] at a cell density of approximately 10⁹ mL⁻¹ and were placed in the Cell Disruption Bomb (Parr Instrument Company). The homogenate produced upon the pressure release was cleared by centrifugations at 4°C, and creatine kinase was added at 0.24 mg.mL ⁻¹ of lysate, before storage in aliquotes at −80°C. Reporter mRNAs were synthesized by transcription in vitro using recombinant T7 RNA polymerase. A non-functional cap (ApppG) (New England Biolabs) was added at the 5′ end of the IRES monocistronic reporter mRNAs to protect them from degradation. Cap-dependent translation was measured with a Renilla Luciferase reporter mRNA that was capped with the ScriptCap m7G capping system (Epicentre Biotechnologies). In vitro translation was performed as previously described (Wakiyama et al., 2005 (link)) and under sub-saturating conditions to avoid substrate titration.

Majzoub K., Hafirassou M.L., Meignin C., Goto A., Marzi S., Fedorova A., Verdier Y., Vinh J., Hoffmann J.A., Martin F., Baumert T.F., Schuster C, & Imler J.L. (2014). RACK1 controls IRES-mediated translation of viruses. Cell, 159(5), 1086-1095.

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Amplified Template Preparation for in vitro Transcription

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All constructs were amplified with a T7 containing forward primer and a T₅₀ tailed reverse primer to prepare templates for in vitro transcription. In vitro transcription and m7G-capping reactions were carried out with T7 RNA Polymerase (NEB) and Vaccinia Capping enzyme (NEB) respectively according to the manufacturer’s protocol. ApppG (NEB) was added to transcription reaction mixture in a proportion of 10:1 to GTP for preparation of A-capped RNAs. m7G-cap and A-capped RNAs were purified with RNA purification kit (Macherey-Nagel).

Ghosh T, & Basu U. (2015). Cis-Acting Sequence Elements and Upstream Open Reading Frame in Mouse Utrophin-A 5'-UTR Repress Cap-Dependent Translation. PLoS ONE, 10(7), e0134809.

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In Vitro Transcription of Capped RNA

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Transcription templates were generated by PCR amplification from the plasmids pUC19-TIE.
The amplified templates were used for in vitro transcription with recombinant T7 RNA polymerase in the presence of either m 7 G ppp G cap analog or non-functional cap analog A ppp G (New England Biolabs®). To check RNA integrity, an aliquot was mixed with Formamide Dye and loaded on 4% denaturing polyacrylamide gel. The RNA is visualized under UV light after ethidium bromide staining. To eliminate unincorporated nucleotides, the RNA sample was loaded on a gel filtration Sephadex G25-column (Pharmacia Fine Chemicals), proteins are then eliminated by phenol extraction and the RNA transcripts are precipitated with 0.25 M NaCl in ethanol. After centrifugation, RNA pellets were dried and resuspended in autoclaved milli-Q water. The concentration of purified RNA samples was determined by absorbance measurement at 260 nm.

Alghoul F., Schaeffer L., Eriani G., & Martin F. (2021). Translation Inhibitory Elements from Hox a3 and a11 mRNAs use uORFs for translation inhibition.

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