PCR products were amplified from extracellular virion DNAs using Easy-A DNA polymerase (Stratagene) and purified using QiaQuick or MinElute PCR purification kits (Qiagen), then either sequenced directly or cloned into plasmids using T/A cloning (Promega) or TOPO XL (Invitrogen) PCR cloning kits. Sanger chain termination sequencing was conducted by either the Biopolymer Laboratory at the University of Maryland at Baltimore or the Nucleic Acids Research Facility at Virginia Commonwealth University. The deletion and the GP89 mutation were confirmed by direct sequencing of R-75 and wild type GPCMV virion DNA.
Topo xl
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TOPO XL is a laboratory equipment product designed for DNA cloning and manipulation. It is a topoisomerase-based cloning system that allows for the rapid and efficient insertion of PCR products into vectors without the need for restriction enzymes or ligase. The TOPO XL system provides a simple and streamlined approach to DNA cloning, making it a valuable tool for researchers in various fields of molecular biology.
Automatically generated - may contain errors
4 protocols using topo xl
1
Extracellular Virion DNA Sequencing
2
Amplifying Disrupted Gene Flanks via Inverse PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Inverse PCR is commonly used to amplify the flanking sequences of the disrupted genes to enable precise determination of the gene’s identity using the UCSC Genome Browser. In this assay, the forward primer binds at just upstream of the polyA signal of the GFP gene and the reverse primer at downstream of the GFP gene, which allows for specific amplification of the flanking sequences of the L1 integration sites.
Digest 1 μg of genomic DNA with Ssp1 restriction enzyme at 37 °C and then purify DNA using a QIAquick PCR purification kit (Qiagen).
Carry out self-ligation of the digested DNA using 20 units of T4 DNA ligase in a total volume of 100 μl.
Use 20 μl (i.e., 200 ng) of the ligation mixture directly as the DNA template for the PCR reaction with forward (5′-TGATAAGATACATTGATGAGTTTGGA-3) and reverse (5′-TATATCTCCCAATGCTATCC-3′) primers in a total volume of 50 μl. Cycling conditions are 1× (94 °C, 2 min), 10× (94 °C, 30 s; 58 °C, 30 s; 72 °C, 1 min), 25× ((94 °C, 20 s; 65 °C, 30 s; 72 °C, 1 min) followed by 1× (72 °C, 7 min).
Clone the amplified PCR product into a Topo-XL (Invitrogen) or pGEM (Promega) vector followed by DNA sequencing using M13 forward and reverse primers.
Identify the flanking sequences of the disrupted regions by BLAST search of the UCSC Mouse Genome Browser.
3
Cloning and Sequencing of AtzDof1.3 Transgene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted as described by Leterrier et al. (2008) [89 (link)]. The A. thaliana transgene was amplified using primers designed to the construct borders flanking the site of AtzDof1.3 transgene LexA: 5′-TACAGTACGTCGAGGGGATGAT-3′ and E9 terminator sequence 5-CTGGTGTGTGCGCAATGAAACTG-3′ of the target vector. The PCR reaction (25 μL) contained 40 ng of DNA, 2× Advantage GC-Melt Buffer, dNTP, LA Polymerase (Clontech, Palo Alto, CA, USA), and 0.4 μM primers. The PCR conditions were as follows: an initial start at 94 °C for 3 min, 30 × cycles of denaturation at 94 °C for 15 s, annealing at 60 °C for 1 min, an extension at 68 °C for 1 min, and a final extension at 68 °C for 5 min. DNA was subcloned into TOPO XL (Invitrogen, Valencia, CA, USA). The nucleotide composition of these fragments was determined using ABI BigDye Terminator sequencing. Gene identification was performed by querying GenBank and the Sol Genomics Network database using the B asic L ocal A lignment S equence T ool (BLAST) [90 (link)]. The promoter sequence was identified using primers designed to the LexA gene 5′-GCCTTCAGATGTTCTTCAGC-3′ as the antisense primer, and phytoene desaturase gene 5′-TAACTGCCAAACCACCACAA-3′ as the sense primer. The PCR reaction was performed using AmpliTaq DNA Polymerase (Applied Biosystems, Waltham, MA, USA) following the protocol exactly as described.
4
Cloning and Sequencing of Rph15 Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Based on the hypersensitive infection type of the Rph15 resistance, we used the physical interval of 44-57Mb reported in Martin et al. (2020) for the introgression of the Rph15 resistance from PI 355447 into Bowman to search for predicted annotated NLR candidate resistance genes in the Morex reference assembly https://apex.ipk-gatersleben.de/apex/f?p=284:10. We designed a single primer pair (RGA4 F: and RGA4 R) based on the Morex rph15 pseudogene sequence (GenBank: AY641411.1) to amplify a 9,442 base pair genomic fragment including approximately: 1.3 kb of sequence upstream from the translation Start codon and 2.5 kb of sequence downstream of the STOP codon. Polymerase chain reaction was performed using Phusion polymerase as per the manufacturer's instructions (New England Biosciences). The PCR products were separated on a 1% agarose gel and a single band of the expected size was excised and purified. The gel-purified PCR product was then cloned into the sub-cloning vector TOPO XL using manufacturer's instructions (Invitrogen). We amplified and cloned genomic fragments from resistant (Bowman+Rph15 and Hs 680) and susceptible (Gus and L94) barley accessions.
The plasmid DNA of five positive clones from each amplicon was sent for Sanger sequencing using internal primers and the sequences were compared with the template from Morex.
The plasmid DNA of five positive clones from each amplicon was sent for Sanger sequencing using internal primers and the sequences were compared with the template from Morex.
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!