Dna isolation kit

We surveyed the cowpea crops for yellow mosaic symptoms at the vegetative growth stage in five states of India, namely, Jharkhand, Chhattisgarh, Uttar Pradesh, Madhya Pradesh and Assam, during 2012–13 [43 –44 ]. The maximum incidence of this viral disease was in a field of Jharkhand state, with a disease incidence of about 60–70%. The cowpea plants in the field exhibited stunted growth, yellow patches, mottling of leaves, reduced leaf size and distortion of leaf lamina symptoms (S1 Fig). The infected plant leaf materials were RCA analyzed, cloned and sequenced. Agroinfectious dimeric clones were prepared for MYMIV cowpea isolate, propagated and maintained in cowpea cultivar Pusa Komal through agroinfiltration and maintained in a greenhouse at 25–27°C. Total genomic DNA was extracted from infected leaf samples by using DNA isolation kit (Hi-Media, Mumbai), the purified genomic DNA were subjected to amplify full length of DNA-A and DNA-B, using TempliPhi^™ DNA-Amplification kit (GE Healthcare, UK) through Rolling circle amplification (RCA) method as per manufacturer’s instructions. The ~2.7 Kb DNA fragments obtained after the restriction digestion (EcoRI, HindIII, BamHI, SacI and EcoRV) of the RCA products were cloned and sequenced. The sequences obtained were analyzed using DNA Star, Mega 5.2, and BIOEDIT version 7.0 programs.