pIRS-21097-1105-specific or pCDC25b38-46-specific murine T-cell lines were magnetically enriched for CD8α (Miltenyi, 130-049-401). Total RNA was isolated using PureLink Micro-to-Midi Total RNA isolation kit (Invitrogen) and cDNA was synthesized using the GeneRacer™ Kit (Life Technologies) as described (33 (link)). 5′RACE PCR was performed using the GeneRacer™ 5′ primer and one of three 3′ gene-specific primers: TCR-CαRev (5′-ACTGGACCACAGCCTCAGCGTCAT-3′), TCR-Cβ1Rev (5′-TGAATTCTTTCTTTTGACCATAGCCAT-3′) or TCR-Cβ2Rev (5′GGAATTTTTTTTCTTGACCATGGCCAT-3′). PCR products of correct size were cloned into the pCR®4-TOPO® vector (Life Technologies). TCR sequences were determined and matched to the IMGT database (34 (link)). TCR sequences are found under GenBank accession nos. KJ542620, KJ542621, KJ542622 and KJ542623.
Purelink micro to midi total rna isolation kit
Manufactured by Thermo Fisher Scientific
The PureLink Micro-to-Midi Total RNA Isolation Kit is a tool designed for the efficient extraction and purification of total RNA from a variety of sample types, including cells, tissues, and microorganisms. The kit utilizes a silica-based membrane technology to capture and purify RNA, enabling the isolation of high-quality RNA for downstream applications.
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Lab products found in correlation
Generacer kit, by Thermo Fisher Scientific (1 mentions)
Pcr4 topo vector, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Faststart universal sybr green master mix, by Roche (1 mentions)
2 protocols using purelink micro to midi total rna isolation kit
1
Murine T-cell Receptor Sequencing
2
RNA Quantification via qPCR
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Total RNA was extracted using the PureLink Micro-to-midi total RNA isolation kit (Invitrogen). Reverse transcription was performed on 500 ng of RNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA). Quantification was performed on a 7900HT fast real-time PCR system (Applied Biosystems) using FastStart Universal Sybr Green master mix (Roche Applied Science, Indianapolis, IN). Primer sequences used are shown in Supplemental Table S1. Results were normalized to β2-microglobulin (B2M) and compared with control samples (2-ΔΔCt method).
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