Preliminary identification of strain identity involved EcoRV restriction enzyme (New England Biolabs, Beverly, MA) of the PCR product produced by the CO1-891F/1472R primers. A strain-specific polymorphism at site 1182 produces in an EcoRV recognition site in the COI-RS haplotype but not with COI-CS [6 , 14 (link), 25 (link)]. Uncut fragments were further analyzed by DNA sequencing to confirm C-strain identity.
Ecorv restriction enzyme
Manufactured by New England Biolabs
Sourced in United Kingdom, United States
EcoRV is a type II restriction enzyme that recognizes and cleaves the palindromic DNA sequence 5'-GATATC-3'. It is commonly used in molecular biology applications, such as DNA cleavage, fragment analysis, and DNA manipulation.
Automatically generated - may contain errors
Lab products found in correlation
Exosap it express pcr product cleanup kit, by Thermo Fisher Scientific (1 mentions)
Platinum superfi 2 dna polymerase, by Thermo Fisher Scientific (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Bamhi restriction enzyme, by New England Biolabs (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Mluci, by New England Biolabs (1 mentions)
Rcutsmart buffer, by New England Biolabs (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
5 protocols using ecorv restriction enzyme
1
Strain Identification via COI Sequencing
2
Genetic Variation Analysis of MARC1 Gene
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DNA from mouse and human livers were extracted by DNeasy Blood & Tissue Kit (Qiagen). DNA area including mouse Marc1 Exon 2 and 3 regions was amplified using Platinum SuperFi II DNA Polymerase (Thermo Fisher) using primers 5′-aattgctgctacctggtgct-3′ and 5′-tggttcatgagggttgtcgg-3′ . PCR product was digested by EcoRV restriction enzyme (New England Biolabs) and separated on 0.8% Agarose gel by electrophoresis. Pair of primers 5′-gctaggagcagcttttctga-3′ and 5′-caacagagccgaggtcatca-3′ were used to amplify whole human MARC1 gene exon 3 by PCR reaction, and ExoSAP-IT Express PCR Product Cleanup Kit (Thermo Scientific) was used to remove both primers before sequencing. Human MARC1 165A/T and 187M/K genetic variants were detected by Sanger sequencing.
3
PCR Product Restriction Digest Analysis
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Six μl of the PCR product was digested with 0.5 μl of the required restriction enzyme in a 20 μl reaction and incubated for 4–8 hrs at 37 °C. For MEN2B HDR templates, the PCR products were digested in BamHI restriction enzyme (New England Biolabs, Ipswich, MA) and in EcoRV restriction enzyme (New England Biolabs, Ipswich, MA) for MEN2A/HSCR HDR experiments. Digested products were then analysed by 1.5% agarose gel electrophoresis. Positive clones were cloned into the pGEM-T Easy vector (Promega, Madison, WI) and cloned products were sequenced.
4
Genotyping P. falciparum Drug Resistance Markers
5
Plasmid Linearization and Extraction
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The plasmid pFA6a-GFP(S65T)-His3MX6 (Longtine et al., 1998 (link)) was a gift from John Pringle (Addgene 41598; Addgene, Watertown, MA, USA) and pFA6a-link-yoTagRFP-T-Kan (Lee et al., 2013 (link)) was a gift from Wendell Lim & Kurt Thorn (Addgene 44906); both were given in the form of bacterial stabs. Note that pFA6a-link-yoTagRFP-T-Kan is the source of the KanR marker used to delete HTB2-HTA2. Five mL of bacteria were cultured overnight, then plasmids were extracted with the QIAprep Spin Miniprep Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions.
Extracted plasmids were linearized by double digestion with a reaction containing 1 µg of plasmid DNA, 5 µL of 10× rCutSmart Buffer (New England BioLabs, Ipswich, MA, USA), 10 units each of SalI and EcoRV restriction enzymes (New England BioLabs) topped up to 50 µL with nuclease-free water. The reaction mixture was heated at 37°C for 15 min for the digestion reaction, then 80°C for 20 min to inactivate the enzymes.
Extracted plasmids were linearized by double digestion with a reaction containing 1 µg of plasmid DNA, 5 µL of 10× rCutSmart Buffer (New England BioLabs, Ipswich, MA, USA), 10 units each of SalI and EcoRV restriction enzymes (New England BioLabs) topped up to 50 µL with nuclease-free water. The reaction mixture was heated at 37°C for 15 min for the digestion reaction, then 80°C for 20 min to inactivate the enzymes.
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