Vector carrying open-reading frame (ORF) of PTEN was a gift from William Sellers74 (link) (pSG5L HA PTEN wt; Addgene#10750). The vector was linearized by ApaI/EcoRI digestion and purified. HA-PTEN ORF under the regulation of T7 promoter was then amplified by PCR reaction. The amplicons were further purified and used as templates for in vitro transcription (IVT). The modified PTEN-mRNA was synthesized as described previously48 (link), 49 (link). In brief, IVT was conducted using MEGAscript T7 kit (Ambion) with 1–2 μg template and 7.5 mM ATP, 1.5 mM GTP, 7.5 mM 5-methyl-CTP, 7.5 mM pseudo-UTP (TriLink Biotechnologies), and 6 mM 3′−0-Me-m7G(5′)ppp(5′)G (anti-reverse cap analog, ARCA) (TriLink Biotechnologies). Reactions were incubated at 37°C for 4 hours, followed by Turbo DNase treatment for 15 min. 3´ poly(A)-tails were further added to IVT RNA products using a poly(A) tailing kit (Ambion). mRNA was purified by using the MEGAclear kit (Ambion), then treated with Antarctic Phosphatase (New England Biolab) at 37°C for 30 min, and further purified. Large-scale PTEN mRNA was custom-prepared by TriLink Biotechnologies as above (ARCA capped and enzymatically polyadenylated; fully substituted with Pseudo-U and 5’-Methyl-C; DNase and phosphatase treatment; Silica membrane purification) using 100–150 μg template containing T7 promoter and HA-PTEN ORF.
Pseudo utp
Manufactured by TriLink
Sourced in United States
Pseudo-UTP is a nucleotide analogue used as a research tool in molecular biology applications. It functions as a nucleotide building block that can be incorporated into nucleic acid sequences.
Automatically generated - may contain errors
Lab products found in correlation
5 methyl ctp, by TriLink (4 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (4 mentions)
Megaclear kit, by Thermo Fisher Scientific (3 mentions)
Antarctic phosphatase, by New England Biolabs (3 mentions)
Poly a tailing kit, by Thermo Fisher Scientific (2 mentions)
Psg5l ha pten wt, by Addgene (2 mentions)
3 0 me m7g 5 ppp 5 g, by TriLink (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Arca cap analog, by New England Biolabs (1 mentions)
B18r interferon inhibitor, by Thermo Fisher Scientific (1 mentions)
Kapa taq kit, by Roche (1 mentions)
3 o me m7g 5 ppp 5 g rna cap structure analog, by New England Biolabs (1 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
T7 flashscribe transcription kit, by CellScript (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rna nano 6000 assay kit, by Agilent Technologies (1 mentions)
Anti reverse cap analog, by TriLink (1 mentions)
Guanosine triphosphate, by TriLink (1 mentions)
6 protocols using pseudo utp
1
Synthesis of PTEN mRNA via in vitro Transcription
2
In Vitro mRNA Synthesis and Transfection in hESCs
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In vitro mRNA synthesis and transfections were performed according to previous protocols.46 (link) In brief, T7 promoter and polyA tail were added by polymerase chain reaction (PCR) using KAPA taq kit (Kapabiosystems, London, UK). RNA was transcribed from the template using MEGAscript T7 kit (Ambion, Carlsbad, CA, USA), with ARCA cap analog (New England Biolabs, Ipswich, MA, USA); ATP; GTP; 5-Methyl-CTP (TriLink, San Diego, CA, USA); and pseudo-UTP (TriLink). Synthesized RNAs were purified with the MEGAclear kit (Ambion). RNA transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. To increase the viability of transfected cells, B18R interferon inhibitor (eBioscience, San Diego, CA, USA) was supplemented to the culture medium. One day before transfection, 30,000 hESCs were seeded on a culture plate, and 1 µg/well of each synthetic modified mRNA was induced. The hESCs were subjected to four consecutive transfections with TF-encoding RNAs, or GFP or mCherry mRNAs as controls with Lipofectamine 2000 (Life Technologies) in StemFit AK-03 with B18R (eBioscience) for the first two days of differentiation. After two consecutive days of transfection, the culture medium was changed to DKSFM with 100 μg/mL CT and 10 ng/mL EGF.
3
In Vitro Transcription and Modified mRNA Synthesis
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After the PCR, the genetic information is in vitro transcribed from DNA to mRNA using MEGAscript® T7 Kit (Life Technologies, Darmstadt, Germany). The generated mRNA transcript is then used to induce protein expression in cells. At first, 23 μl NTP/cap analog mixture containing 7.5 mM ATP, 1.875 mM GTP (both from MEGAscript® T7 Kit), 7.5 mM Me-CTP, 7.5 mM Pseudo-UTP (both from TriLink BioTechnologies, San Diego, USA), and 2.5 mM 3′-O-Me-m7G(5′)ppp(5′)G RNA cap structure analog (New England Biolabs, Frankfurt am Main, Germany) was prepared and mixed thoroughly. The IVT reaction mixture of 40 μl was then assembled by adding 40 U RiboLock RNase inhibitor (Thermo Scientific, Waltham, USA), 1 μg PCR product, 1× reaction buffer and 1× T7 RNA polymerase enzyme mix. The IVT reaction mixture was incubated at 37°C for 3 h in a thermomixer. To remove the template DNA, 1 μl TURBO DNase (from MEGAscript® T7 Kit) was added to the IVT reaction mixture and incubated for 15 min at 37°C. Then, the reaction mixture was purified using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. The modified mRNA was eluted from the spin column membrane twice with 40 μl nuclease-free water.
4
Synthesis of PTEN mRNA via in vitro Transcription
5
Comprehensive MPXV Protein Characterization
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All mRNA sequences encoding MPXV proteins (A29L, A35R, M1R, B6R) were prepared by in vitro transcription, as described previously.22 (link) Transcription was performed from linearized DNA templates using the T7-FlashScribe™ Transcription Kit (Cellscript). Further, the modified mRNA was synthesized by replacing UTP in the kit with pseudo-UTP (TriLink). Afterwards, the RNA was capped using the ScriptCap™ Cap 1 Capping System kit with ScriptCap™ Capping enzyme and 2'-O-methyltransferase (Cellscript) according to the manufacturer’s instructions. The mRNA product purified by ammonium acetate precipitation was then resuspended in RNase-free water for further analysis and application.
The concentration and quality of the synthesized MPXV mRNA were measured using an Agilent 2100 Bioanalyzer and RNA Nano 6000 Assay Kit (Agilent), according to the manufacturer’s instructions.
All mRNAs (2 μg) were transfected into HEK293T cells using Lipofectamine 3000 transfection reagent (Thermo Fisher) according to the manufacturer’s instructions, lysates were collected, and Western blotting was performed.
The concentration and quality of the synthesized MPXV mRNA were measured using an Agilent 2100 Bioanalyzer and RNA Nano 6000 Assay Kit (Agilent), according to the manufacturer’s instructions.
All mRNAs (2 μg) were transfected into HEK293T cells using Lipofectamine 3000 transfection reagent (Thermo Fisher) according to the manufacturer’s instructions, lysates were collected, and Western blotting was performed.
6
Synthesis of mRNA and miR-switches
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mRNAs and miR-switches were synthesized as previously described (Miki et al., 2015 (link); Warren et al., 2010 (link)). Briefly, they were generated with a MEGAscript T7 Transcription Kit (Ambion, cat. no. AMB13345) and a modified protocol. Template DNAs, T7 enzyme, ATP, guanosine triphosphate, pseudo-UTP (Tri-Link Bio Technologies, cat. no. N-1019-10), 5-methyl-CTP (Tri-Link Bio Technologies, cat. no. N-1014-10) and Anti Reverse Cap Analog (Tri-Link Bio Technologies, cat. no. N-7003-10) were reacted at 37 °C for 4 h. Adding TURBO DNase, the reacted products were further incubated at 37 °C for 30 min. The resulting mRNAs and miR-switches were incubated with Antarctic Phosphatase (New England Biolabs, cat. no. M0289S) at 37 °C for 30 min. The RNeasy MinElute Cleanup Kit (QIAGEN, cat. no. 74204) was used to purify the mRNAs and miR-switches.
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