Total cellular RNA was extracted from clonal hybridomas that produced MARV antibodies, and RT-PCR reaction was performed using mixtures of primers designed to amplify all heavy chain or light chain antibody variable regions. The generated PCR products were purified and cloned into the pJet 1.2 plasmid vector (Thermo Scientific, #K1231) for sequence analysis. The nucleotide sequences of plasmid DNAs were determined using an ABI3700 automated DNA sequencer. Heavy chain or light chain antibody variable region sequences were analyzed using the IMGT/V-Quest program (Brochet et al., 2008 (link); Giudicelli et al., 2011 (link)). The analysis involved the identification of germline genes that were used for antibody production, location of complementary determining regions (CDRs) and framework regions (FRs) as well as the number and location of somatic mutations that occurred during affinity maturation.
Pjet1.2 plasmid vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PJET1.2 plasmid vector is a DNA cloning vector used for the insertion and expression of target genes in bacterial cells. It has a size of 2.9 kilobase pairs and contains essential genetic elements such as an origin of replication and an antibiotic resistance marker for selection purposes.
Automatically generated - may contain errors
4 protocols using pjet1.2 plasmid vector
1
Antibody Variable Region Sequencing and Analysis
2
Metagenomic Discovery of a Fish Polyomavirus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RCA DNA (20 µl aliquot from each sample) was sequenced on an Illumina 4000 (Illumina) sequencer at Macrogen (Korea). The paired-end reads were de novo assembled using ABySS v2.02 [29 (link)], and contigs >250 nucleotides were analysed using blast x [30 (link)] against a local viral database. A contig of 372 nucleotides was identified in the liver sample with similarities to VP2 and VP1 of fish PyVs. Based on the sequence of this contig, a pair of abutting primers (F: 5′-CGCTGCTAAAGGAAATAAAATCAAGATATGGGAAT-3′; R: 5′-ACTGGAATTTTAGGCAAATCTATAGCTGACGTTAG-3′) were designed to recover the full genome of this putative PyV. A 0.5 µl sample of the RCA reaction was used as a template for PCR amplification using the abutting primers and Kapa HiFi Hotstart DNA polymerase with the following thermal cycling protocol: 95 °C for 3 min; 25 cycles of 98 °C for 20 s, 60 °C for 15 s, 72 °C for 5 min, and a final extension of 72 °C for 6 min. The amplicon was resolved on a 0.7 % agarose gel, the ~5 kb product was excised, gel-purified and cloned into the pJET1.2 plasmid vector (ThermoFisher, USA). The recombinant plasmid was Sanger-sequenced by primer walking at Macrogen (Korea), and the Sanger sequence contigs were assembled using DNA Baser V4 (Heracle BioSoft S.R.L., Romania).
3
Circovirus Genome Identification in Fecal Samples
4
Isolation and Sequencing of TYLCV Genomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total DNA was extracted from tomato samples displaying leaf curl symptoms from Australia (n ¼52), Iran (n¼ 12) and Mauritius (n ¼6). Circular DNA was enriched by rolling circle amplification (RCA) using Templiphi (GE Healthcare, USA). Unit length TYLCV genomes were recovered from the RCA concatemers using XmnI, NcoI, BamHI or SalI restriction enzymes, and cloned into pJET 1.2 plasmid vector (ThermoFisher, USA) for XmnI digested genomes and into pBluescript SK (Stratagen, USA) for NcoI, BamHI or SalI digested genomes. The recombinant plasmids were Sanger sequenced by primer walking at Macrogen Inc. (South Korea). Complete genome sequences were assembled using DNA Baser V4 (Heracle Biosoft S.R.L., Romania).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!