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Incidence, distribution and characteristics of major tomato leaf curl and mosaic virus diseases (Chiemsombat et al., 1992; Nakhla et al., 1998). Each primer had to be specific to a particular annealing region of the viral DNA genome as depicted in Figure 3.9 and 3.11. 3.2.2.2.2.3 Agarose Gel Electrophoresis The PCR DNA product extracted above was then run in 1% agarose gel electrophoresis in TBE (Tris-borate-EDTA) buffer, 0.5% water, to determine successful amplification of the desired DNA fragment. DNA was stained with bromophenol blue using 2 µl of dye to 6 µl of PCR DNA product. A total of 8 µl was dispensed to each mini-gel well and run at 95 - 119 volts for ca. 15 - 20 min. Gels were then stained with ethidium bromide, 0.5 µl/ml, for 5 min and destained in water for 5 min (Czosnek et al., 1988; Nakhla et al., 1998). Stained gel was viewed under Ultra Violet light and photos taken with a digital computerized camera, Pharmacia Biotech (Teare, 1996). 3.2.2.2.3 Cloning and Sequencing Using DNA amplified in PCR experiments, cloning which is the multiplication of DNA in a plasmid vector (Lewin, 1997) was done to purify DNA prior to sequencing. Cloning was done for isolates IG1, K1 and RL5, which had reacted positively to geminivirus degenerate primers. Primers used in cloning included PAL1v 1978 and PAR1c 715, PARAv 494 and PARAc 1048, and specific primers PTYCRv 21 and PTYCRc 287, which are also used to test for genus Begomovirus in the western hemisphere. TOPO®- TA cloning kit protocol was used (Promega, 1996) for cloning. Plasmid pCR 2.1 TOPO® vector was used in a ligation mixture of 0.5 – 2 µl of fresh PCR product, 4 µl of disodium water and 1 µl of the vector. One Shot® bacterial cells of Escherichia coli were used as cloning agents in 2 µl of 0.5M β-mercaptoethanol and 250 µl of SOC medium (Invitrogen, 1998) or 1 x YT medium as substitute (Nakhla, personal communication, 1998). The One Shot® bacterial cells of E. coli were transformed by exposure to ice cold (4 ºC) temperatures for 30 min; heat shock for 30 seconds at 42 ºC, and ice cooling for 2 min. The mixture was incubated at 37 ºC on a horizontal rotor for 30 min. 66
PTYAL1c 2196 PAL1v 1978 Incidence, distribution and characteristics of major tomato leaf curl and mosaic virus diseases PTYC 2v 1499 PTYCRv21 PTYCRc 287 PARAv 494 PARAc 1048 Figure 3.7: Different primers used in molecular analysis experiments and approximate points where they start annealling on a single stranded DNA (ssDNA) genome of TYLCV, and in reverse or forward direction depending on the DNA section to be amplified. 67 PARIc 715
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Incidence, Distribution and Charact
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Charles SSEKYEWA Incidence, Distrib
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CONTENTS 1 CHAPTER 1...............
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ACKNOWLEDGEMENT The following perso
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vii 7 mosaic virus (CMV), Alfalfa m
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ix 9 Consequently, our findings con
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SAMENVATTING Tomaat is een economis
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xiii 13 Beide genhomologie percenta
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xv 15 dit onderzoek geven ook aan d
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LIST OF TABLES xvii 17 Table Page 1
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ACRONYMS AND ABBREVIATIONS xix 19 A
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Abbreviation Word in full PCR Polym
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Incidence, distribution and charact
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Table 2.1: The orthography of some
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Page 126 and 127: 1200 1200 (A) IG1 (ToLCV-UG) PAL1v
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A A C D Incidence, distribution and
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Opaque and not smeared Translucent
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Rainfall (cm) Temp. Cº Mean Whitef
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8 ANNEXES Incidence, distribution a
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Table A.1.1 continued Incidence, di
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Annex 5: Incidence, distribution an
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Annex 6: Incidence, distribution an
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