Pgem t easy vector kit

Amplification of the partial mtCO1 fragment was performed using forward primer 2195Bt (5ʹ-TGRTTTTTTGGTCATCCRGAAGT-3ʹ) and reverse primer C012/Bt-sh2 (5ʹ-TTTACTGCACTTTCTGCC-3ʹ) (Mugerwa et al. 2018 ). PCR reaction mixtures (20µL) contained 10µL of 2 × reSource™ Taq Mix (reSource Taq DNA Polymerase, 6 mM MgCl₂, 2 mM dNTPs) (Source BioScience, UK), 1µL of each 10 µM primer, 6µL of molecular biology-grade water (Sigma-Aldrich) and 2µL of DNA template. Initial denaturation (94 °C 2 min) was followed by 35 cycles of denaturation (94 °C, 20 s), primer annealing (52 °C, 30 s) and extension (72 °C, 1 min). A final extension (72 °C, 10 min) was performed before storing reactions at 4 °C. Electrophoresis of PCR products was on 2%(w/v) agarose gels in 0.5 × TBE stained with RedSafe™ (iNtRON Biotechnology, Korea). PCR products were visualised under UV light (302 nm) and those of the expected size (864 bp) purified for sequencing and cloning using a reSource™ PCR purification kit (Source BioScience, UK). Purified PCR products were sent for Sanger sequencing (Source BioScience, UK). Where a novel sequence was identified, purified PCR products were cloned from three separate PCR reactions using the pGEM®-T easy vector kit (Promega, UK) and resequenced to confirm the novel sequence. Sequences generated were deposited in GenBank (accession numbers MK444227-MK445130).

A 5’RACE was performed using the 5’ RACE System for Rapid Amplification of cDNA Ends, version 2.0 kit (Invitrogen, USA). Briefly, cDNA was generated by reverse transcription from total RNA extract followed by degradation of the RNA. dC-tailing was then performed with the cDNA and the resulting dC-tailed DNA was used as the template in PCR as described in the kit instructions. The PCR products were analyzed by agarose gel electrophoresis (1% agarose in TAE buffer). To identify the transcription start sites, PCR products were inserted into the pGEM-T easy vector kit as described by the manufacturer (Promega, USA). Insert were then PCR-amplified and the resulting PCR products were sequenced.

The biosynthetic precursor cDNA of SL-BBI was obtained as previously described [25 (link)]. Briefly, the Dynabeads^® mRNA DIRECT^TM Kit (Dynal Biotech, Merseyside, UK) was used to isolate the mRNA from the lyophilised skin secretion. The first-strand cDNA was synthesised by the BD SMART^TM RACE cDNA Amplification Kit (BD Bioscience Clontech, UK) and used to construct the cDNA library through 3′ RACE-PCR. For SL-BBI, the 3′-RACE reactions employed a NUP primer (supplied with the kit) and a degenerate sense primer (3′-RACE: 5′-GAWYYAYYHRAGCCYAAADATGTTCA-3′, R = A/G, V = A/C/G, N = A/C/T/G, Y = C/T, S = C/G, W = A/T) was designed based on the highly conserved nucleic acid sequences of the 5′-untranslated region of the protease inhibitory peptide published previously. The purified products were cloned by pGEM^®-T Easy Vector Kit (Promega Corporation, Madison WI, USA) and sequenced through an ABI3730 automated sequencer (Applied Biosystems, Foster City, CA, USA).