mRNA transcripts of Tlr1, 2 and 6 were produced as previously described [17 (link)]. In brief, T7-promoter-containing pVAX.A120-vectors encoding for Tlr1, 2 and 6 were linearized and transcribed in vitro into chemically modified mRNA, incorporating 25% 2-Thio-UTP and 25% 5-Methyl-CTP (TriLink Bio Technologies) using the T7 MEGAscript kit (Ambion). Modified mRNA was purified using the MEGAclear kit (Ambion) and dissolved in RNase-free DEPC-water.
5 methyl ctp
Manufactured by TriLink
Sourced in United States
5-methyl-CTP is a laboratory reagent used in molecular biology and biochemistry research. It is a modified form of the nucleotide cytosine triphosphate (CTP). The methyl group at the 5-position of the cytosine ring is the core functional modification of this compound.
Automatically generated - may contain errors
Lab products found in correlation
Megaclear kit, by Thermo Fisher Scientific (4 mentions)
Pseudo utp, by TriLink (4 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (4 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)
2 thio utp, by TriLink (1 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)
Hiscribe t7 arca mrna kit with tailing, by New England Biolabs (1 mentions)
Pcr purification kit, by Qiagen (1 mentions)
Anti reverse cap analog, by TriLink (1 mentions)
Guanosine triphosphate, by TriLink (1 mentions)
Adenosine triphosphate (atp), by TriLink (1 mentions)
Megascript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
6 protocols using 5 methyl ctp
1
Production of Modified mRNA for Tlr1, 2, 6
2
Synthesis of PTEN mRNA via in vitro Transcription
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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.
3
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.
4
In Vitro Transcription of mRNAs
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To prepare template DNA for IVT, each DNA template including the transcription cassette was amplified by PCR. The amplicons were purified using a PCR purification kit (Qiagen) before being used as templates for IVT. In vitro transcribed mRNAs were prepared using a HiScribe T7 ARCA mRNA kit (with tailing) (NEB) with 0.5–1 μg template DNA and 1.25 mM of 5-methyl-CTP (TriLink Biotechnologies). Reaction conditions and mRNA purification procedures followed the NEB manufacturer’s instructions without modification.
5
Synthesis of PTEN mRNA via in vitro Transcription
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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