Plant Virology Protocols. From Virus Isolation to Transgenic by Ron S. S. Fraser

By Ron S. S. Fraser

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6+ 61 1 D: neutral _ __ _ _ * 62 Covey et al double-stranded DNAs, dtscontmuous double-stranded DNAs, open circular, supercoiled, and hairpin molecules (22,22). After electrophorests and before blotting, a depurmatton step IS mcluded to introduce breaks mto molecules, which would otherwise renature by snapback on neutraltzatton The duration/acid concentration of the treatment IS fairly critical and wtll depend upon gel thlckness A balance has to be attained between undertreatment, resultrng tn underrepresentation of snapback forms on the final autoradlogram, and overtreatment, causing a general reductron m hybrldtzatton signal of all forms References 1 Howell, S H (1985) The molecular biology of plant DNA vnuses CRC Crzt Rev Plant Scz 2,287-3 16 2 Covey, S N and Hull, R (1992) Genetic engineering with double-stranded DNA vtruses, m Genetzc Engzneerrng wzth Plant &uses (Davies, J W and Wtlson, T M A , eds ), CRC, Boca Raton, p 217 3 Franck, A , Gutlley, H , Jonard, G , Richards, K , and Hnth, L (1980) Nucleottde sequence of cauliflower mosaic virus DNA Cell 21,285-294 4 Gardner, R C , Howarth, A J , Hahn, P, Brownluedt, M , Shepherd, R J , and Messing, J (198 1) The complete nucleottde sequence of an infectious clone of caultflower mosaic vu-us by Ml 3mp7 shotgun sequencing.

26) reported sets of prtmers useful for amplification of parts of the genome of all Curto- and Begomoviruses. If the PCR products are cloned and sequenced, one can then design abutting or partially overlapping PCR primers that will facilitate amplification and clonmg of an entire genomic component of the virus of interest (see refs. 27-29). 2. 1. 1. 5 Waring-type blender Cheesecloth. 2. lM EDTA, pH 7 0, autoclaved. After autoclavmg, add SDS to 1% (w/v) 2 Trts-buffered phenol, prepared as described m Sambrook et al (31).

27. Rice Dwarf Virus 1 Harvest l&50 g fresh race leaves 2 Homogenize the leaves with ELISA jurce press (Erich Pollahne, Germany) m three to five times (v/w) of 0 IA4 phosphate buffer 3 Add 1% (w/v) Drrselase whrle starring at 6°C wrth a magnetrc strrrer for 1 h. 4 Add one-third vol of chloroform, mix the extract thoroughly for 3 mm wrth Polytron homogemzer on ice 5 Centrifuge at 3000g for 15 mm 6 Transfer the aqueous phase to a centrrfuge tube, leavmg Interface Centrrfuge at 62,000g in a Hrtachr RP 30-2 rotor for 60 mm at 4’C 7.

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