Extraction of total RNA from a mix of 10 complete R. antilleana first to fifth instar nymphs was performed using Trizol. cDNA was synthesized using SuperScript III First-Strand kit according to the manufacturer's instructions (Invitrogen). Fragments of Scr and Ubx genes were amplified (electronic supplementary material, figures S1 and S2) by PCR using GoTaq G2 DNA polymerase (Promega) and the primers described in electronic supplementary material, table S1. PCR products were examined by electrophoresis, purified using PCR Minelute kit (Qiagen) and cloned into a pGEM-T vector kit (Promega). The sequences of R. antilleana Scr and Ubx can be retrieved in GenBank using the following accession numbers: MG999826 and MG999808 , respectively, and in electronic supplementary material, figures S1 and S2.
Pgem t vector kit
Manufactured by Promega
The pGEM-T vector kit is a commercially available plasmid vector used for the cloning and amplification of DNA fragments. The vector is supplied with T4 DNA ligase and buffer, allowing for the efficient ligation of DNA inserts. The pGEM-T vector contains T-overhangs on the 3' ends, facilitating the direct insertion of PCR products. The vector also includes an ampicillin resistance gene for selection and a multiple cloning site for the insertion of DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Gotaq g2 dna polymerase, by Promega (1 mentions)
Superscript 3 first strand kit, by Thermo Fisher Scientific (1 mentions)
Minelute pcr kit, by Qiagen (1 mentions)
3130xl genetic analyser, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
2 protocols using pgem t vector kit
1
Molecular characterization of Scr and Ubx in R. antilleana
2
Rabbit Kidney Cell Line Sequence Analysis
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For all primers (exon spinning, listed in Table 2) RT-PCR products were purified and cloned into the pGEM-T vector (pGEM-T Vector Kit, Promega GmbH). Recombinant plasmids were sequenced using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems/Thermo Fisher Scientific). Sequence analysis was performed with 3130xl Genetic Analyser (Applied Biosystems/Thermo Fisher Scientific) in the Core Facility: DNA-Sequencing of the Medical Faculty (Halle, Germany). Fragment identity was proven by BLASTN and BLASTX tool.A new partial LC3 (194 bp) and mTOR (249 bp) cDNA was amplified from the rabbit kidney cell line RK13 using human primers. The sequence was 95% for mTOR and 97% for LC3 identical with the human one at the mRNA level, as shown by BLAST tool alignment. Experiments with same number: animals were handled in parallel regarding mating and embryo collection at day 6 post coitum.
Enhanced mTORC1 signalling in diabetic pregnancy
Enhanced mTORC1 signalling in diabetic pregnancy
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