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Computer-simulated virtual RFLP patterns generated from in silico digestions of phytoplasma 16S rRNA genes from strains detected in this study belonging to groups: (A) 16SrI using HhaI (MH428961: 16SrI-P *); (B) 16SrIII using BstUI (MH428959: 16SrIII-F *); (C) 16SrIX using RsaI (MH428962:16SrIX-A[infinito]); (D) 16SrXIII using MseI (MH428958: 16SrXIII-H *); (E, F) 16SrXV using HaeIII and HpalI (MH428963: 16SrXV-A[infinito]), obtained using iPhyClassifier tool.
For RFLP, the URA5 gene amplified product was subjected to double enzymatic digestion using the Sau96I (5000 U/mL) and HhaI (20000 U/mL) enzymes (New England BioLabs, USA) incubated at 37[degrees]C in dry for 3 hours.
The 54Thr polymorphism abolishes the HhaI enzyme restriction site resulting in un-cut 180bp band.
Los cebadores amplifican un fragmento de 557 pb del gen gmk y despues de la digestion con la enzima HhaI, se generan fragmentos de 429 y 124 pb para el ST5 mientras que para el ST8 se generan fragmentos de 323, 124, y 105 pb.
Restriction endonuclease analysis using HhaI and HpaII to discriminate among group B Pasteurella multocida associated with haemorrhagic septicaemia.
Kod genotipa e2/e2 javljaju se fragmenti od 91 bp i 83 bp jer HhaI ne sece GTGC region cisteinskih rezidua.
Restriction endonuclease analysis for characterization of serotypes of hemorrhagic septicaemia can be done with the enzyme HhaI. Discrimination of the isolates can be done by application of ribotyping as well as large DNA separation by means of pulsed-field gel electrophoresis.
Following the supplier's instructions, aliquots of 5-|mL amplified PCR products were digested with 2 U HhaI restriction enzyme (MBI Fermentas, Germany) at 37[degrees]C for 10 h.
Two (mu) L of each amplicon were digested with EcoRI, AluI, HhaI and TaqI endonucleases, according to the manufacturers" instruction (Promega, USA) at 37degC (65degC for TaqI) overnight.
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- Hh protein
- HH Vohwinkel
- HHa5 hair keratin type-I intermediate filament